Inhibition of polyomavirus replication

ABSTRACT

The invention relates to antisense molecules and methods for modulating splicing of polyomavirus T antigen pre-mRNA. In one aspect the invention relates to an antisense oligonucleotide 12 to 30, preferably 17, 18, 19 or 20 to 30 nucleobases in length which comprises a sequence that is the reverse complement of a contiguous stretch of at least 12 nucleobases of a polyomavirus T-antigen pre-mRNA and which antisense oligonucleotide can modulate splicing of said T-antigen pre-mRNA in a cell.

RELATED APPLICATIONS

This application is a division of U.S. Pat. Application Serial No.16/977,779, filed Sep. 2, 2020, which is a 35 U.S.C. §371 filing ofInternational Patent Application No. PCT/NL2019/050131, filed Mar. 1,2019, which claims priority to European Patent Application No.18159797.2, filed Mar. 2, 2018, the entire disclosures of which arehereby incorporated herein by reference.

SUBMISSION OF SEQUENCE LISTING

The contents of the electronic submission of the text file SequenceListing, which is titled 730897_LUMC9-001USDIV_ST.txt, which was createdon Jun. 22, 2022 and is 18.2 KB in size, is incorporated herein byreference in its entirety.

The invention relates to molecules that specifically bind topolyomavirus RNA. In some embodiments molecules comprise antisenseoligonucleotides that recognize pre-mRNA with a coding region of a Tantigen of a polyomavirus. Examples of polyomaviruses are the humanpolyomaviruses such as JC virus (JCV), BK virus (BKV) or Merkel cellvirus (MCV).

Polyomaviruses are small non-enveloped double-stranded DNA viruses whosenatural hosts are normally mammals and birds. Infections in adults aremostly asymptomatic but can become pathological when the immune systemis compromised. Non-limiting examples of human polyomaviruses are BKvirus, JC virus and Merkel cell virus.

JCV and BKV are both opportunistic pathogens which infect the humanpopulation during early childhood (Leploeg, M.D. et. al., ClinicalInfectious Diseases, 2001). The sero-prevalence in adults is high. Bothviruses are thought to remain latent in kidney cells of the host(Wunderink, H.F. et. al., American Journal of Transplantation, 2017).Reactivation can occur, for instance, in immunosuppressed individuals(Wunderink, H.F. et. al., American Journal of Transplantation, 2017;Parajuli, S. et. al., Clinical Transplantation, 2018; Gard, L. et. al.,PLoS One, 2017).

Polyomaviruses share a common genome structure. They have genes that areexpressed both early and late in the infection cycle. Both early andlate genes produce RNAs from which through differential splicing,various proteins can be stranslated. As shown in FIG. 1 , the late RNAstypically encode the three capsid proteins whereas the early genes codefor the small and large T-antigens and often one or more otheralternatively spliced coding regions (Helle, F. et. al., Viruses, 2017).

WO2015/042466 describes an antisense oligonucleotide-based approach toinhibit JC virus replication and multiplication. The antisenseoligonucleotides disclosed therein can be eitheroligodeoxyribonucleotides (ODNs) or be chimeric oligonucleotides thathave an ODN interior flanked by one or more nucleotides with a nucleaseresistant backbone. The latter render an RNA:oligonucleotide hybridsensitive to the action of RNaseH. The deoxyribonucleotides interior hasat least 4 deoxyribonucleotides and is flanked by nuclease resistantregions that have 2′-sugar-modified nucleotides. The antisenseoligonucleotides are directed towards specific sequences that arepresent in JC virus mRNA.

In some embodiments, the present invention provides antisenseoligonucleotides that can modulate splicing of a polyomavirus T-antigenpre-mRNA. Antisense oligonucleotides may have a sequence that iscomplementary to a splice donor site and/or a splice acceptor site insaid pre-mRNA. Antisense oligonucleotides may have a sequence that iscomplementary to one or more exon-adjacent intron nucleobases (see FIG.2 ). In some embodiments the antisense oligonucleotide renders a duplexof the antisense oligonucleotide with its polyomavirus T-antigenpre-mRNA target resistant to the action of RNaseH.

SUMMARY OF THE INVENTION

The invention provides an antisense oligonucleotide 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length which comprises a sequencethat is the reverse complement of a contiguous stretch of at least 12nucleobases of a polyomavirus large T-antigen pre-mRNA and whichantisense oligonucleotide can modulate splicing of said large T-antigenpre-mRNA in a cell (see FIG. 1 ).

Splice modulating antisense oligonucleotides of the present inventiontypically require a contiguous stretch of at least 17, preferably atleast 18, more preferably at least 19 and more preferably at least 20nucleobases complementary to the polyomavirus large T-antigen pre-mRNA.

The invention also provides an antisense oligonucleotide 12 to 30,preferably 17, 18, 19 or 20 to 30 nucleobases in length which comprisesa sequence that is the reverse complement of a contiguous stretch of atleast 12 nucleobases of a polyomavirus large T-antigen pre-mRNA, whichstretch comprises a splice donor, a splice acceptor sequence or acombination thereof in said pre-mRNA. The splice donor or acceptorsequence is preferably a polyomavirus large T antigen splice acceptor ora polyomavirus large T antigen splice donor sequence.

Further provided is an antisense oligonucleotide12 to 30, preferably 17,18, 19 or 20 to 30 nucleobases in length, that is at least 80%complementary to nucleotides 4537- 4596 or 4881-4940 taken fromNC_001538 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4397-4456 or the region4741-4800 taken from NC_001699 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4299-4358 or the region4686-4745 taken from NC_009238 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4477-4536 or the region4876-4935 taken from NC_009539 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4693-4752 or the region5124-5183 taken from NC_010277 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4264-4323 or the region4654-4713 taken from NC_014406 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4272-4331 or the region4677-4736 taken from NC_014407 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4352-4411 or the region4765-4824 taken from NC_014361 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4408-4467 or the region4760-4819 taken from NC_015150 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4303-4362 or the region4658-4717 taken from NC_018102 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4159-4218 or the region4504-4563 taken from NC_020106 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4392-4451 or the region4791-4850 taken from NC_020890 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

Also provided is an antisense oligonucleotide of 12 to 30, preferably17, 18, 19 or 20 to 30 nucleobases in length that is preferably at least80% complementary to nucleotides in the region 4471-4530 or the region4859-4918 taken from NC_024118 and at least comprising complementarityto the splice donor or splice acceptor sequence in the respectiveregions.

An antisense oligonucleotide as described herein preferably comprises atleast 12 contiguous nucleobases of SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ IDNO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQID NO: 18; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23;SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27, whereinthe at least twelve nucleotides preferably comprise the reversecomplement of the splice donor site, splice acceptor site or combinationthereof of the target pre-mRNA, i.e. the splice donor/acceptor of thelarge T-antigen pre-mRNA of the respective polyomavirus. In a preferredembodiment the antisense oligonucleotide as described herein comprisesat least 17, preferably at least 18, preferably at least 19 and morepreferably at least 20 contiguous nucleobases of SEQ ID NO: 1; SEQ IDNO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ IDNO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ IDNO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQID NO: 17; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22;SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO:27; preferably SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4;SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10;SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO:24 or SEQ ID NO: 25; preferably SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO:3; SEQ ID NO: 4; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO:23; SEQ ID NO: 24 or SEQ ID NO: 25, wherein the nucleotides preferablycomprise the reverse complement of the splice donor site, spliceacceptor site or combination thereof of the target pre-mRNA, i.e. thesplice donor/acceptor of the large T-antigen pre-mRNA of the respectivepolyomavirus. An antisense oligonucleotide may have one mismatch withthe indicated sequence, the mismatch is not at the start or the end ofthe contiguous stretch as an AON with a contiguous stretch of 17nucleotides, for instance, with a mismatch at position one or 17 wouldactually have a contiguous stretch of 16 nucleotides.

Also provided is a method of inhibiting polyomavirus replication in acell, the method comprising providing a cell that is infected with saidpolyomavirus with an antisense oligonucleotide of the invention that isspecific for the polyomavirus.

The invention further provides a method of preparing a graft fortransplantation, the method characterized in that donor cells,preferably donor kidney cells are provided with an antisenseoligonucleotide of the invention that is specific for the polyomavirus.

Also provided is a method of treatment of a polyomavirus infection in asubject, the method comprising administering an antisenseoligonucleotide of the invention that is specific for the polyomavirus,to the individual in need thereof.

Further provided is a method of administering an antisenseoligonucleotide to an individual, for hybridization to a complementaryRNA sequence in a cell of said individual, the method characterized inthat the antisense oligonucleotide is a chimeric antisenseoligonucleotide comprising a first and a second region, and wherein saidfirst region comprises one or more deoxyribonucleotides and said secondregion comprises at least one 2′-O-(2-methoxy-ethyl) nucleotide (seeFIG. 7 ).

Further provided is a method of inhibiting replication of a polyomavirusin a cell, the method comprising providing said cell with an antisenseoligonucleotide 12 to 30 nucleotides in length which comprises asequence that is the reverse complement of a contiguous stretch of atleast 12 nucleobases of a polyomavirus large T-antigen pre-mRNA andwhich antisense oligonucleotide can modulate the splicing of said largeT-antigen pre-mRNA. Said antisense oligonucleotide is preferably anantisense oligonucleotide as described herein, preferably an antisenseoligonucleotide of SEQ ID NO: 1-25 as described or modified herein.

DETAILED DESCRIPTION OF THE INVENTION

The term ″antisense oligonucleotide′ refers to an oligomer or polymer ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or mimetics,chimeras, analogs and homologs thereof. This term includes antisenseoligonucleotides composed of naturally occurring nucleobases, sugars,and covalent internucleoside (backbone) linkages as well as antisenseoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted antisense oligonucleotides areoften preferred over native forms because of desirable properties suchas, for example, enhanced cellular uptake, enhanced affinity for atarget nucleic acid, and increased stability in the presence ofnucleases (see FIG. 7 ).

An antisense oligonucleotide as described herein is preferably asingle-stranded antisense oligonucleotide.

Antisense oligonucleotides of the present invention also includemodified antisense oligonucleotides in which a different base is presentat one or more of the nucleotide positions in the antisenseoligonucleotide. For example, if the first nucleotide is an adenosine,modified antisense oligonucleotides may be produced that containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the antisense oligonucleotide. These antisenseoligonucleotides are then tested using the methods described herein todetermine their ability to inhibit T-antigen RNA.

An antisense oligonucleotide of the present invention can hybridize topolyomavirus RNA produced upon infection of a susceptible cell. As such,the antisense oligonucleotide comprises a sequence that is the reversecomplement of the sequence of (the part of) the target RNA. Theantisense oligonucleotide may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compound. In a preferred embodiment the antisenseoligonucleotide is a single stranded antisense oligonucleotide.

The antisense oligonucleotide may be linked to one or more otherchemical structures. The other structure may be a peptide or protein, asugar, a lipid or other chemical structure. The other structure may alsobe one or more other nucleotides. The one or more other nucleotides mayperform a function different from the antisense part. For instance,hybridization to another nucleic target sequence. The other structuremay perform any of a number of one or more different functions.Non-limiting examples of such functions are stability of the antisenseoligonucleotide, increase in bioavailability, increase in cellpenetration, increase in nuclear delivery, targeting to specific cellsand the like.

Once introduced into a system, the antisense oligonucleotides of theinvention may elicit the action of one or more enzymes or structuralproteins to effect modification of the target nucleic acid. It is knownin the art that single-stranded antisense oligonucleotides that are“DNA-like’ elicit RNase H, a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target. This is one way to enhance the efficiencyof antisense oligonucleotide-mediated inhibition of gene expression. Inembodiments antisense oligonucleotides of the invention do not, and arenot designed to, recruit the action of RNase H to the targetRNA/antisense oligonucleotide hybrid. Similar roles have been postulatedfor other ribonucleases such as those in the RNase III and ribonucleaseL family of enzymes. Antisense oligonucleotides as described hereinpreferably have modifications that confer nuclease resistance to theantisense oligonucleotide and the target RNA/antisense oligonucleotidehybrid. Specifically excluded from the definition of “antisenseoligonucleotides” herein are ribozymes that contain internal or external“bulges” that do not hybridize to the target sequence.

An antisense oligonucleotide in accordance with the invention comprisesof 12 to 30, preferably 17, 18, 19 or 20 to 30 nucleobases (i.e. ofabout 12 to and including 30 linked nucleosides). One of ordinary skillin the art will appreciate that the invention embodies antisenseoligonucleotides of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 nucleobases in length. One having ordinaryskill in the art will appreciate that this embodies antisenseoligonucleotides of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleobases in length. One having ordinary skill in theart will appreciate that this embodies antisense oligonucleotides of 17,18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleobases in length. Oftenantisense oligonucleotides are 18, 19, 20, 21 or 22 nucleobases inlength. Antisense oligonucleotides as described herein are preferably17, 18, 19, 20, 21, or 22 nucleotides in length.

In one embodiment an antisense oligonucleotide comprises at least 12contiguous nucleobases of a sequence of an oligonucleotide of which thesequence is specifically disclosed herein. Antisense oligonucleotides12-30 nucleobases in length comprising a stretch of at least twelve (12)consecutive nucleobases selected from within the illustrative antisenseoligonucleotides are considered to be suitable antisenseoligonucleotides as well.

An antisense oligonucleotide comprises a sequence of nucleobases that isthe reverse complement of the sequence of the target RNA. An antisenseoligonucleotide as described herein comprises a stretch of at least 12nucleobases with a sequence that is the reverse complement of thesequence of at least 12 contiguous nucleobases of the target RNA. Thestretch is also referred to as the complementarity region or thehybridization region. One of ordinary skill in the art will appreciatethat the invention embodies antisense oligonucleotides with acomplementarity region of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length. One of ordinaryskill in the art will appreciate that the invention embodies antisenseoligonucleotides with a complementarity region of 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length. Oneof ordinary skill in the art will appreciate that the invention embodiesantisense oligonucleotides with a complementarity region of 17, 18, 19,20, 21, 22, 23, 24, 25 or 26 nucleobases in length. Often antisenseoligonucleotides have a complementarity region of 18, 19, 20, 21 or 22nucleobases in length. Antisense oligonucleotides as described hereinpreferably have a complementarity region of 17, 18, 19, 20, 21 or 22nucleobases in length.

Antisense oligonucleotides preferably have a length that is commensurateto the length of the complementarity region.

The target sequence of an antisense oligonucleotide as described hereinis a part of a polyomavirus T-antigen pre-mRNA. Pre-mRNA or precursormRNA is an immature single strand of messenger ribonucleic acid (mRNA).Polyomavirus T-antigen pre-mRNA is synthesized from a polyomavirus DNAtemplate in the cell nucleus by transcription. The pre-mRNA contains oneor more introns that are spliced out during maturation of the pre-mRNAinto mRNA. The splicing process removes introns from transcripts andjoins exons together. Introns are typically flanked by a donor site (5′end of the intron) and an acceptor site (3′ end of the intron). Thesplice sites are required for splicing and typically include an almostinvariant sequence GU at the 5′ end of the intron and a splice acceptorsite at the 3′ end of the intron with an almost invariant AG sequence.The GU and AG sequence and the intervening sequence are spliced out ofthe pre-mRNA. A characteristic of polyomavirus T-antigen pre-mRNA isthat it can be alternatively spliced or not spliced leading to thegeneration of at least two and often 3, 4 or 5 differently spliced mRNAs(see FIG. 1 ). Virus propagation is dependent on the availability of thevirus genome, the presence of virus proteins, the cellular machinery andparticularly the delicate interplay between the various stages andcomponents. The splicing process of virus RNAs is an important methodfor regulating the virus propagation process, and influences the leveland likely also the timing of certain products being formed in thecells. In the present invention it was surprisingly found that directingan oligonucleotide of the invention to a splice donor or splice acceptorsite for splicing of large T-antigen mRNA has a profound effect not onlyon the production of T-antigen mRNA, but also on the level capsidprotein produced and the production of virus by the infected cell.

The target sequence of an antisense oligonucleotide as described hereincomprises a splice donor site of a polyomavirus T-antigen pre-mRNA, asplice acceptor site of a polyomavirus T-antigen pre-mRNA or acombination thereof. The target sequence typically comprises a stretchof 12 contiguous nucleobases comprising a splice donor site of apolyomavirus T-antigen pre-mRNA or a splice acceptor site of apolyomavirus T-antigen pre-mRNA. The contiguous sequence preferablycomprises an intron sequence in addition to the splice donor or thesplice acceptor sequence. In a preferred embodiment the contiguoussequence comprises one or two intron nucleobases adjacent to the splicedonor or the splice acceptor sequence. In a preferred embodiment thecontiguous sequence comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 intronnucleobases adjacent to the splice donor or splice acceptor sequence. Ina preferred embodiment the contiguous sequence comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, or 28 exon nucleobases adjacent to the splice donor or thesplice acceptor sequence. The antisense oligonucleotide preferablycomprises a sequence that is the reverse complement of a contiguoussequence of nucleobases of the target pre-mRNA. The sum of the number ofintron, splice site and exon nucleobases does not exceed the totalnumber of nucleobases in the antisense oligonucleotide.

The target sequence preferably comprises the splice donor sequence orthe splice acceptor sequence that define the intron that is otherwisespliced out to produce mRNA that codes for the large T-antigen of thespecific polyomavirus (see FIG. 8 ).

The sequence of an antisense oligonucleotide as described hereincomprises complementarity to the splice donor sequence or the spliceacceptor sequence that define the intron that is otherwise spliced outto produce mRNA that codes for the large T-antigen of the specificpolyomavirus (see FIG. 8 ).

In a preferred embodiment the target sequence of a first oligonucleotideis a sequence in the 3′ splice site target region indicated below forthe respective viruses. In a preferred embodiment the target sequence ofanother oligonucleotide is a sequence in the 5′ splice site targetregion indicated below for the respective viruses. An oligonucleotidethat is directed towards a target sequence in a region indicated belowcomprises the complementary sequence of the splice donor or spliceacceptor in the indicated sequence. If more than one AON is used in amethod as described herein it is preferred that the AONs are directedtowards a target sequence of the same virus.

Abbreviation Accession 3′ splice site target region 5′ splice sitetarget region BKPyV NC_001538 4537-4596 4881-4940 JCPyV NC_0016994397-4456 4741-4800 KIPyV NC_009238 4299-4358 4686-4745 WUPyV NC_0095394477-4536 4876-4935 MCPyV NC_010277 4693-4752 5124-5183 HPyV6 NC_0144064264-4323 4654-4713 HPyV7 NC_014407 4272-4331 4677-4736 TSPyV NC_0143614352-4411 4765-4824 HPyV9 NC_015150 4408-4467 4760-4819 MWPyV NC–0181024303-4362 4658-4717 STLPyV NC_020106 4159-4218 4504-4563 HPyV12NC_020890 4392-4451 4791-4850 NJPyV NC_024118 4471-4530 4859-4918

The first column contains an abbreviation of the virus name. A prototypesequence for the virus is indicated with the accession code for thesequence in the sequence database. The third and fourth columnsspecifies a region in the prototype virus sequence that contains thesplice donor (column 4) or splice acceptor (column 3) to which an AON asdescribed herein can comprise a complementarity region.

An antisense oligonucleotide according to the invention may modulate thesplicing of T-antigen pre-mRNA in the infected cell. Without being boundby theory it is believed that an antisense oligonucleotide as describedherein inhibits usage of the splice site it is targeted to. Theresultant reduced production of large T-antigen impacts the expressionof the capsid proteins and thereby the production of virus (see FIGS.4-6 ). Without being bound by theory it is believed that the imbalanceof T-antigen specific splice products induced by the antisenseoligonucleotides of the invention has a more pronounced effect on viruspropagation than the reduction of T-antigen mRNA specific mRNA byRNAi-like approaches.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of nucleic acid. In the present invention, thepreferred mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,adenine and thymine are complementary nucleobases that pair through theformation of hydrogen bonds. Hybridization can occur under varyingcircumstances. Hybridization of complementary strands typically improveswith the length of the sequence. Specific hybridization of two strandsis accomplished with a contiguous stretch of 12 or more complementarynucleobases. The sequence of an antisense oligonucleotide can be, butneed not necessarily be, 100% complementary to that of its targetsequence to be specifically hybridizable. Moreover, an antisenseoligonucleotide may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent. In one embodiment of this invention, the antisenseoligonucleotide of the present invention comprises at least 70%, or atleast 75%, or at least 80%, or at least 85% sequence complementarity toa target region within the target pre-mRNA. In other embodiments, theantisense oligonucleotide of the present invention comprises at least90% sequence complementarity and even comprise at least 95% or at least96% sequence complementarity to the target region within the targetpre-mRNA. For example, an antisense compound in which 18 of 20nucleobases of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. When an antisense oligonucleotide of 18nucleotides has a sequence that is the reverse complement of acontiguous stretch of at least 12 nucleobases of a polyomavirus largeT-antigen pre-mRNA, the remaining 6 complementary nucleobases may beclustered with the 12 or not be contiguous with the 12. Percentcomplementarity of an antisense oligonucleotide with a region of atarget pre-mRNA can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschulet al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7,649-656). As the number of nucleotides is always aninteger, the actual percentage may be not be exactly 90% or not exactly96%. The contiguous sequence preferably has no or only one nucleotidemismatch with the target nucleic acid sequence.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4537- 4596 or the region 4881-4940 taken fromNC_001538 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4537-4596 or 4881-4940 taken fromNC_001538. The 12 to 30, preferably 17, 18, 19 or 20 to 30 nucleobasesare preferably complementary to a stretch of at least 12, 13, 14, 15,16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30 contiguousnucleotides of the target pre-mRNA. In a preferred embodiment theoligonucleotide is 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases in length and preferably comprises a sequence as set forthin SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5;SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO:24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27; preferably as setforth in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ IDNO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ IDNO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24 orSEQ ID NO: 25; preferably as set forth in SEQ ID NO: 1; SEQ ID NO: 2;SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22;SEQ ID NO: 23; SEQ ID NO: 24 or SEQ ID NO: 25.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4397-4456 or the region 4741-4800 taken fromNC_001699 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4397-4456 or the region 4741-4800 takenfrom NC_001699. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4299-4358 or the region 4686-4745 taken fromNC_009238 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4299-4358 or the region 4686-4745 takenfrom NC_009238. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4477-4536 or the region 4876-4935 taken fromNC_009539 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4477-4536 or the region 4876-4935 takenfrom NC_009539. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4693-4752 or the region 5124-5183 taken fromNC_010277 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4693-4752 or the region 5124-5183 takenfrom NC_010277. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4264-4323 or the region 4654-4713 taken fromNC_014406 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4264-4323 or the region 4654-4713 takenfrom NC_014406. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4272-4331 or the region 4677-4736 taken fromNC_014407 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4272-4331 or the region 4677-4736 takenfrom NC_014407. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4352-4411 or the region 4765-4824 taken fromNC_014361 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4352-4411 or the region 4765-4824 takenfrom NC_014361. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4408-4467 or the region 4760-4819 taken fromNC_015150 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4408-4467 or the region 4760-4819 takenfrom NC_015150. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4303-4362 or the region 4658-4717 taken fromNC_018102 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4303-4362 or the region 4658-4717 takenfrom NC_018102. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4159-4218 or the region 4504-4563 taken fromNC_020106 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4159-4218 or the region 4504-4563 takenfrom NC_020106. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4392-4451 or the region 4791-4850 taken fromNC_020890 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4392-4451 or the region 4791-4850 takenfrom NC_020890. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide of 12 to 30, preferably 17, 18, 19 or 20 to30 nucleobases in length is preferably at least 80% complementary tonucleotides in the region 4471-4530 or the region 4859-4918 taken fromNC_024118 and at least comprising complementarity to the splice donor orsplice acceptor sequence in the respective regions, preferably at least90% complementary to nucleotides 4471-4530 or the region 4859-4918 takenfrom NC_024118. The 12 to 30, preferably 17, 18, 19 or 20 to 30nucleobases are preferably complementary to a stretch of at least 12,13, 14, 15, 16, 17, 18, 19, 30, 21, 22, 23 ,24, 25 ,26 ,27 ,28, 29 or 30contiguous nucleotides of the target pre-mRNA.

An antisense oligonucleotide as described herein preferably comprises atleast 12 contiguous nucleobases of SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ IDNO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQID NO: 18; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23;SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27 preferablyof SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6;SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 20;SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24 or SEQ ID NO:25; preferably of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO:4; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ IDNO: 24 or SEQ ID NO: 25, wherein the at least twelve nucleotidespreferably comprise the reverse complement of the splice donor site,splice acceptor site or combination thereof of the target pre-mRNA, i.e.the splice donor/acceptor of the large T-antigen pre-mRNA of therespective polyomavirus. The reverse complement is present in therespective SEQ IDs.

The invention further relates to an antisense oligonucleotide comprisingat least 12 contiguous nucleobases of the nucleotide sequence (see FIG.3 and FIG. 10 ):

5′ ACCUCUGAGCUACUCCAGGU 3′ SEQ ID NO: 1;5′ ACAAACCUCUGAGCUACUCC 3′ SEQ ID NO: 2;5′ CAGCACAAACCUCUGAGCUA 3′ SEQ ID NO: 3;5′ UCCAUAGGUUGGCACCUAGA 3′ SEQ ID NO: 4;5′ UGUUCCAUAGGUUGGCACCU 3′ SEQ ID NO: 5;5′ AAACCUCUGAGCUACUCCAG 3′ SEQ ID NO: 20;5′ GCACAAACCUCUGAGCUACU 3′ SEQ ID NO: 21;5′ AUCAGCACAAACCUCUGAGC 3′ SEQ ID NO: 22;5′ AAAUCAGCACAAACCUCUGA 3′ SEQ ID NO: 23;5′ GAAAAUCAGCACAAACCUCU 3′ SEQ ID NO: 24;5′ AGGAAAAUCAGCACAAACCU 3′ SEQ ID NO: 25;5′ CAUAGGUUGGCACCUAUAAA 3′ SEQ ID NO: 26 or5′ UUCCAUAGGUUGGCACCUAU 3′ SEQ ID NO: 27.

An antisense oligonucleotide as described herein preferably comprises atleast 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of SEQ IDNO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ IDNO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ IDNO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 21;SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO:26 or SEQ ID NO: 27; preferably of SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ IDNO: 9; SEQ ID NO: 10; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQID NO: 23; SEQ ID NO: 24 or SEQ ID NO: 25; preferably of SEQ ID NO: 1;SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 20; SEQ ID NO: 21;SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24 or SEQ ID NO: 25, whereinthe at least respectively 13, 14, 15, 16, 17, 18, 19 or 20 nucleotidespreferably comprise the reverse complement of the splice donor site,splice acceptor site or combination thereof of the target pre-mRNA. Anucleobase as indicated herein may be substituted by a differentnucleobase with same base pairing activity in kind not necessarily inamount. An example of such an alternative is the base thymidine as asubstitute for uracil. Other nucleobases may be substituted for analternative with the same kind of base pairing activity.

Another alternative are bases that pair with any base. An example of abase is inosine. Such bases do typically not add specificity to theoligonucleotide compared to a base that pairs with the appropriate basein the target RNA. Oligonucleotides can accommodate this to some extentas is known to the person skilled in the art. If the same specificity isdesired an additional selective base can be added to theoligonucleotide, for instance but not limited to one additionalselective base for each inosine or other non-selective base.

The antisense oligonucleotide preferably comprises at least 12contiguous nucleobases of the nucleotide sequence (see FIG. 9 ):

SEQ ID Virus Target Sequence 1 BKPyV Donor 5′ ACCUCUGAGCUACUCCAGGU 3′ 2BKPyV Donor 5′ ACAAACCUCUGAGCUACUCC 3′ 3 BKPyV Donor 5′CAGCACAAACCUCUGAGCUA 3′ 4 BKPyV Acceptor 5′ UCCAUAGGUUGGCACCUAGA 3′ 5BKPyV Acceptor 5′ UGUUCCAUAGGUUGGCACCU 3′ 6 JCPyV Donor 5′ACCUCUGAACUAUUCCAUGU 3′ 7 JCPyV Donor 5′ ACCAACCUCUGAACUAUUCC 3′ 8 JCPyVDonor 5′ CACAACCAACCUCUGAACUA 3′ 9 JCPyV Acceptor 5′UCCAUAGGUUGGCACCUAAA 3′ 10 JCPyV Acceptor 5′ UGUUCCAUAGGUUGGCACCU 3′ 11KiPyV Donor 5′ GUAUACCUGAGAAGAUUGCC 3′ 12 KiPyV Donor 5′UCUUUGCAGUAUACCUGAGA 3′ 13 KiPyV Acceptor 5′ UGUACCGUAUGUAGGUAUCU 3′ 14KiPyV Acceptor 5′ CCGUAUGUAGGUAUCUAUAC 3′ 15 WUPyV Donor 5′UCUACCUGUGAAGAGCUCCA 3′ 16 WUPyV Donor 5′ UGUGCAUUCUACCUGUGAAG 3′ 17MCPyV Donor 5′ CCUCAUCAAACAUAGAGAAG 3′ 18 MCPyV Donor 5′GGAAAUUUUGUACUGACCUC 3′ 19 Control 5′ AGGUCCACACUCAAUCCUCA 3′ 20 HYB_06Donor 5′ AAACCUCUGAGCUACUCCAG 3′ 21 HYB_07 Donor 5′ GCACAAACCUCUGAGCUACU3′ 22 HYB_08 Donor 5′ AUCAGCACAAACCUCUGAGC 3′ 23 HYB_09 Donor 5′AAAUCAGCACAAACCUCUGA 3′ 24 HYB_10 Donor 5′ GAAAAUCAGCACAAACCUCU 3′ 25HYB_11 Donor 5′ AGGAAAAUCAGCACAAACCU 3′ 26 HYB_12 Acceptor 5′CAUAGGUUGGCACCUAUAAA 3′ 27 HYB_13 Acceptor 5′ UUCCAUAGGUUGGCACCUAU 3′ 28HYB_14 Coding Exon 1 5′ UGAGCUCCAUGGAUUCUUCC 3′ 29 SAN-73 Coding Exon 25′ CACTCTTCTGTTCCAT 3′ 30 SAN-74 Donor 5′ CACAAACCTCTGAGCT 3′

In one embodiment, the antisense oligonucleotide as described hereincomprises at least one backbone modification. In one embodimentantisense oligonucleotide comprises a phosphorothioate modification. Thephosphorothioate (PS) modification substitutes a sulfur atom for anon-bridging oxygen in the phosphate backbone of an oligo. Thismodification renders the internucleotide linkage resistant to nucleasedegradation. Phosphorothioate bonds can be introduced between the last 3to 5 nucleotides at the 5′- or 3′-end of the oligo to inhibitexonuclease degradation (see FIG. 7 ). Including phosphorothioate bondsthroughout the entire oligo will help reduce attack by endonucleases aswell (see FIG. 3 ).

In another embodiment the antisense oligonucleotide comprises amorpholino (phosphorodiamidate morpholino) modification. Whilemorpholinos have standard nucleic acid bases, those bases are bound tomorpholine rings linked to each other by phosphorodiamidate groupsinstead of phosphates. Morpholinos do not trigger the degradation oftheir target RNA molecules.

In one embodiment, the antisense oligonucleotide comprises at least onesugar modification on the 2′ carbon of the ribose moiety of thenucleoside. In one embodiment, the antisense oligonucleotide comprisesat least one 2′ sugar modification. An overview of sugar modificationsfor anti-sense purposes is given in Prakash (2011; Chem. Biodivers. Sept8(9): 1616-1641. Doi 10.1002/cbdv.201100081). As shown in FIG. 7 ,preferred 2′ sugar modifications are 2′- alkoxy or 2′-alkoxyalkoxymodifications, more preferably 2′-methoxyethoxy. Preferred modificationsare 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE),2′-S-constrained-ethyl (2′-cEt) and locked nucleic acid (LNA).

Other modifications include peptide nucleic acid (PNA).

In some embodiments all positions in a given antisense oligonucleotideare uniformly modified. In other embodiments some positions in a givenantisense oligonucleotide are not uniformly modified. In fact, more thanone of the aforementioned modifications may be incorporated in a singleantisense oligonucleotide or even in a single nucleoside within anantisense oligonucleotide. The present invention also includes antisenseoligonucleotides which are chimeric antisense oligonucleotide. Chimericantisense oligonucleotides are antisense oligonucleotides which containtwo or more chemically distinct regions, each made up of at least onenucleotide. These antisense oligonucleotides contain at least one regionwherein the antisense oligonucleotide is modified so as to confer uponthe antisense oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid.

An antisense oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. Often such a possibilityis mediated by the inclusion of a region in the interior of theantisense oligonucleotide. Such antisense oligonucleotides are alsoreferred to as “gapmers”. A gapmer is a chimeric antisenseoligonucleotide that contains a typically central block ofdeoxyribonucleotides monomers sufficiently long to induce RNaseHcleavage. Efficient RNase cleavage requires a stretch of 4 or moredeoxyribonucleotides. Typically such stretches have 9 or moredeoxyribonucleotides. In the present invention it is preferred that theantisense oligonucleotide does not contain a region that can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.

The antisense oligonucleotide preferably comprises modified nucleotidessuch as phosphorothioate- modified nucleobases and/or 2′ sugarmodifications thereby providing resistance to inadvertent degradation bynucleases. As shown in FIG. 7 , preferred 2′ sugar modification are 2′-alkoxy or 2′-alkoxyalkoxy modifications, more preferably2′-methoxyethoxy. Preferred 2′ sugar modifications include 2′-O-methyl(2′-O-Me), 2′-O-methoxyethyl (2′-MOE), 2′-S-constrained-ethyl (2′-cEt)and locked nucleic acid (LNA). The backbone may contain phosphorothioatethroughout. Other configurations of antisense oligonucleotide are alsocomprehended by this invention. The antisense oligonucleotide preferablycomprises modified nucleotides providing resistance to inadvertentdegradation by nucleases of the target RNA, such as phosphorothioatemodified nucleobases and/or 2′ sugar modifications. Such an antisenseoligonucleotide is within the scope of the invention. An oligonucleotideas disclosed herein thus preferably has a region that provides nucleaseresistant to the target RNA. The duplex formed by the antisenseoligonucleotide and the target RNA is not sensitive to the action ofRNase H.

In a preferred embodiment the antisense oligonucleotide comprises a oneor more nucleobases with a modified polymer backbone. The modifiedpolymer backbone is preferably a modified backbone is indicatedelsewhere herein. In a preferred embodiment the modified polymerbackbone comprises a 2′-O-methyl (2′-0-Me), 2′-O-methoxyethyl (2′-MOE),2′-S-constrained-ethyl (2′-cEt), locked nucleic acid (LNA), peptidenucleic acid (PNA) or morpholino (PMO) nucleotide. In a preferredembodiment all of the nucleotides in the modified polymer backbonecomprises a modified sugar moiety, preferably at the 2′ carbon. In apreferred embodiment the phosphate group linking two nucleotides ismodified into a phosphorothioate group. Preferably all of thephosphodiester linkages are phosphorothioate linkages in the modifiedpolymer backbone.

The human polyomaviruses can be divided into several genera, referred toas the alpha, the beta and the delta genus (Helle, F. et. al., Viruses,2017). In a preferred embodiment the polyomavirus is an alpha or a betavirus, preferably a beta virus. Several human polyomaviruses are listedin the table herein below. In a preferred embodiment the polyomavirus isa BK polyomavirus (or BK virus, also referred to in this application asBKPyV or BKV), a JC polyomavirus (or JC virus, also referred to in thisapplication as JCV) or a Merkel cell polyomavirus (MC polyomavirus, MCvirus, or also referred to in this application as MCV). In aparticularly preferred embodiment the polyomavirus is BK virus or JCvirus, preferably BK virus.

Abbreviation Accession 3′ splice site target region 5′ splice sitetarget region BKPyV NC_001538 4537-4596 4881-4940 JCPyV NC_0016994397-4456 4741-4800 KIPyV NC_009238 4299-4358 4686-4745 WUPyV NC_0095394477-4536 4876-4935 MCPyV NC_010277 4693-4752 5124-5183 HPyV6 NC_0144064264-4323 4654-4713 HPyV7 NC_014407 4272-4331 4677-4736 TSPyV NC_0143614352-4411 4765-4824 HPyV9 NC_015150 4408-4467 4760-4819 MWPyV NC_0181024303-4362 4658-4717 STLPyV NC_020106 4159-4218 4504-4563 HPyV12NC_020890 4392-4451 4791-4850 NJPyV NC_024118 4471-4530 4859-4918

The invention also provides a method of inhibiting polyomavirusreplication in a cell, the method comprising providing a cell that isinfected with said polyomavirus with the antisense oligonucleotide asdescribed herein. The antisense oligonucleotide is preferably anoligonucleotide that targets the polyomavirus of the infection (see FIG.3 ). The cell is preferably a cell that is susceptible for replicationof the polyomavirus. When the cell is a cell in an animal such as ahuman, it is preferred that the animal is a permissive host, i.e. a hostthat allows a virus to circumvent its defenses and replicate the virus.For polyomaviruses, such as BK virus and JC virus, replication is oftendetected first by detecting viruses in the urine of the animal (uremia).Later, when the infection persists, virus can also be detected in theserum of the animal (viremia). Most humans encounter BK and JC virusduring childhood, causing mild illness. However, when reactivated oracquired in the immunocompromised host, BK and JC virus have beenimplicated in a number of human clinical disease states. BK is mostcommonly associated with renal involvement, such as ureteral stenosis,hemorrhagic cystitis and nephropathy (Leploeg, M.D. et. al., ClinicalInfectious Diseases, 2001; Helle, F. et. al., Viruses, 2017).Susceptibility or permissiveness of the host can be induced bycompromising the hosts immune system. Various circumstances can lead toa temporary or permanent reduction of the hosts immune capability.Immunodeficiency (or immune deficiency) is a state in which the immunesystem’s ability to fight infectious disease and cancer is compromisedor entirely absent. Most cases of immunodeficiency are acquired(“secondary”) due to extrinsic factors that affect the patient’s immunesystem. Examples of these extrinsic factors include HIV infection,extremes of age, and environmental factors, such as nutrition.Immunosuppression can also be induced by some drugs, such asglucocorticoids, cytostatics, antibodies, and compounds that act uponimmunophilins (such as calcineurin inhibitors, belatacept (animmunoglobulin like molecule that has the extracellular domain ofCTLA-4) and similar molecules). This can be a desired effect such as inorgan transplant surgery as an anti-rejection measure and in patientssuffering from an overactive immune system, as in autoimmune diseases.However, sometimes this desired effect has the additional effect ofreducing the individuals ability to combat virus infections, such aspolyomavirus infection. A person who has an immunodeficiency of any kindis said to be immune-compromised. An immunocompromised person may beparticularly vulnerable to opportunistic infections, in addition tonormal infections that could affect everyone.

In one embodiment the invention provides a method of preparing a graftfor transplantation, the method characterized in that donor cells,preferably donor kidney cells are provided with the antisenseoligonucleotide as described herein. The antisense oligonucleotide ispreferably an oligonucleotide that targets a polyomavirus thatreplicates in the graft cells. In the case of a kidney graft or kidneycell graft the polyomavirus is preferably a BK virus, a JC virus, or aMC virus-specific oligonucleotide. The cells of the graft thus treatedare less susceptible to replication of the polyomavirus that theantisense oligonucleotide is specific for. This increases the successrate of the transplant. It facilitates the management of transplantrecipients. One of the ways to manage opportunistic polyomavirusreplication in transplant recipients and other drug-induced immunesuppression in patients is to reduce the administration of theimmunosuppressive drug, thereby allowing the immune system to recover tothe extent that the infection and or replication of the virus isreduced. When a graft is prepared as described herein, the polyomavirusinfections/replication in a patient is less frequent and, when detected,often less severe when compared to patients receiving untreated grafts.It is preferred that graft recipients receive one or more additionaladministrations with the antisense oligonucleotide as desired. Theinvention also provides a method of treatment of a polyomavirusinfection in a subject, the method comprising administering theantisense oligonucleotide as described herein, to the individual in needthereof. The individual is preferably an immune-compromised individual.

The graft is preferably an allograft or a xenograft. Recipients of suchgrafts are often treated with immunosuppressive drugs to increase thesurvival of the graft, or to decrease the incidence and/or the severityof host versus graft effects. Many tissues can presently be grafted.Host versus graft effects are often a high risk when transplanting cellsor organs from another, non-genetically identical human or a non-humananimal. Grafts include lung, heart, heart valve, kidney, liver,pancreas, intestine, thymus and bone marrow. Polyomaviruses have beendetected in the plasma of up to 3% of these patients receivingimmunosuppression following organ transplantation (De Vlaminck, I. et.al., Cell, 2013). The graft preferably comprises a kidney, or kidneycells. The individual is preferably the recipient of a kidney or kidneycell transplant.

The antisense oligonucleotide is preferably one that confers resistanceto RNase H to a duplex of the oligonucleotide and the target RNA. Theantisense oligonucleotide preferably comprises a sequence that is thereverse complement of a contiguous stretch of at least 12 and preferablyat least 17 nucleobases of an RNA that can be present in human kidneycells, preferably an RNA of a human virus that can replicate in humankidney cells, preferably a polyomavirus.

In one embodiment the invention provides an antisense oligonucleotide 12to 30, preferably 17, 18, 19 or 20 to 30 nucleobases in length whichcomprises a sequence that is the reverse complement of a contiguousstretch of at least 12 nucleobases of a polyomavirus T-antigen pre-mRNAand which antisense oligonucleotide can modulate splicing of saidT-antigen pre-mRNA in a cell, wherein the antisense oligonucleotidecomprises at least 12 contiguous nucleobases of SEQ ID NO: 1; SEQ ID NO:2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7;SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12;SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO:17; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ IDNO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27.

Further provided is an antisense oligonucleotide comprising at least 12contiguous nucleobases of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ IDNO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18;SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO:24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27, wherein the at leasttwelve nucleotides comprise the reverse complement of the splice donorsequence or the splice acceptor sequence of the large T-antigen pre-mRNAof the respective polyomavirus.

The antisense oligonucleotide as described herein preferably comprises amodification that renders the mRNA-oligonucleotide duplex resistant tothe action of RNase H. Preferably comprising at least one nucleobasewith a modified polymer backbone, preferably a 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-S-constrained-ethyl (2′-cEt), lockednucleic acid (LNA), peptide nucleic acid (PNA) or morpholino (PMO)nucleotide. Preferably all of the nucleobases comprise a modifiedpolymer backbone, preferably a 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl(2′-MOE), 2′-S-constrained-ethyl (2′-cEt), locked nucleic acid (LNA),peptide nucleic acid (PNA) or morpholino (PMO) nucleotide.

In a preferred embodiment the antisense oligonucleotide comprises atleast 17, preferably at least 18, 19 and preferably at least 20contiguous nucleobases of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ IDNO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18;SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO:24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27 or comprising at least17, preferably at least 18, 19 and preferably at least 20 contiguousnucleobases of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4;SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9;SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO:14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ IDNO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27 with one mismatch and whereinthe mismatch is not the first or the last nucleotide of the contiguousstretch.

Also provided is a method of inhibiting polyomavirus replication in acell, the method comprising providing a cell that is infected with saidpolyomavirus with the antisense oligonucleotide. Also provided is amethod of preparing a graft for transplantation, the methodcharacterized in that donor cells, preferably donor kidney cells areprovided with the antisense oligonucleotide.

Further provided is a method of treatment of a polyomavirus infection ina subject, the method comprising administering the antisenseoligonucleotide as described herein, to the individual in need thereof.Said individual is an immune-compromised individual. The individual ispreferably the recipient of a kidney or kidney cell transplant.

Also provided is a method of administering an antisense oligonucleotideto an individual, for hybridization to a complementary RNA sequence in akidney cell of said individual, the method characterized in that theantisense oligonucleotide is an oligonucleotide as described herein.

Further provided is an antisense oligonucleotide comprising amodification that renders a duplex of the antisense oligonucleotide andthe target mRNA resistant to the action of RNase wherein the antisenseoligonucleotide comprises the sequence of SEQ ID NO: 1; SEQ ID NO: 2;SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7;SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12;SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO:17; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ IDNO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26 or SEQ ID NO: 27,with no, one or two mismatches.

It has been observed that oligonucleotides as described herein areefficiently delivered to and taken up by the relevant kidneys cells whenadministered to the animal. The method of administration is preferablyIV administration. For polyomaviruses that can cross the blood-brainbarrier into the central nervous system (CNS) such as JC-virus it ispossible to administer an antisense oligonucleotide to the CNS. Variousways are known in the art. For antisense oligonucleotide mediatedtreatment of JC virus infection of the CNS intrathecal delivery ispreferred. JC virus infection can also be combatted by IV delivery asthe initial infection is thought to be often via the tonsils orgastro-intestinal tract whereupon it spreads to other organs such as butnot limited to tubular epithelial cells in the kidneys where it mayremain latent or continue to reproduce, shedding virus particles in theurine and the brain.

The antisense oligonucleotide preferably does not have a sequence thatconsists of the sequence

5′-CACAAACCTCTGAGCTA (SEQ ID NO: 31);5′-AACCUCUGAACUAUUCCAUGU (SEQ ID NO: 32);5′-ACCUCUGAACUAUUCCAUGUA (SEQ ID NO: 33);5′-TTCATCTGTTCCATAGGTTGGCACCTA (SEQ ID NO: 34); or5′-TTCCATAGGTTGGCACCTAAAAAAAAA (SEQ ID NO: 35), or an alternativethereof where one or more thymidine’s are uracil’s and vice versa.

For the purpose of clarity and a concise description features aredescribed herein as part of the same or separate embodiments, however,it will be appreciated that the scope of the invention may includeembodiments having combinations of all or some of the featuresdescribed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Schematic of BK viral genome and sequence similarity of variousBK virus genome subtypes. Left: The BK virus genome encodes five primaryproteins, namely small t and large T antigen (early genes), as well asagnoprotein and the major capsid proteins VP1, VP2 and VP3 (late genes).The viral genome also contains a non-coding region (NCCR) which containsboth the origin of replication, as well as the promoter region fortranscription factors that drive the expression of the early and lategenes, along with viral genome replication. Of note, alternativesplicing of early and late pre-mRNAs results in numerous proteinisoforms, including small t antigen, truncated T antigen (2 introns) andlarge T antigen (1 intron), along with alternative splicing determiningthe proportion of VP1, VP2 or VP3 that is generated. However, it isimportant to note that the primary splice variant generated for the lateregion is the mature mRNA that encodes predominantly VP1. Continued: Thegenomic sequence of the early region encodes the T antigen proteins,namely small t (tAg), truncated T (truncTAg) and large T antigen (TAg).tAg plays a critical role in driving infected cells into S phase,allowing for TAg-mediated viral genome replication. Furthermore, TAgbinds to the NCCR to drive expression of the late region pre-mRNA. Thisleads to the production of VP1, VP2 and VP3, which are essential forencapsulation of the viral DNA.

FIG. 2 : Whole-gene sequences of TAg, including intron sequences, werealigned using clustalW (“msa” package in R) for 245 uniqueBK-polyomavirus isolates (downloaded from the publicly available NCBIdatabase). From these records, only the isolates reporting a completegenome were used for the conservation of the splice sites in TAg. TheDunlop strain was used as a reference genome. A phylogenetic tree wasconstructed using the UPGMA method (“phangorn” and “ggtree” packages inR). A sequence logo was constructed for the acceptor and donor splicesites to show nucleotide specific conservation between subtypes (“msa”package in R).

FIG. 3 : Composition of antisense oligonucleotides to modulate BKPyV TAgsplicing. Sequences of antisense oligonucleotides (AONs) directedtowards the exon 1 – intron junction (AONs #1, #2 and #3) and intron –exon 2 junction (AONs #4 and #5) of BKPyV TAg.

FIG. 4 : TAg splice-modulating AONs reduce expression levels of TAg andVP1 mRNAs in BKPyV infected human epithelial cells. Left: Reduction inTAg RNA levels in scramble AON-treated HK2 cells versus HK2 cellstreated with splice-modulating AONs #1, #2, #3, #4 and #5 at 7 daysfollowing infection with BKPyV virus at a multiplicity of infection of~100 (n=5; p<0.05). Right: Reduction in VP1 RNA levels in scrambleAON-treated HK2 cells versus HK2 cells treated with splice-modulatingAONs #1, #2, #3, #4 and #5 following infection with BKPyV virus at amultiplicity of infection of ~100 (n=5; p<0.05).

FIG. 5 : TAg splice-modulating AONs reduce expression levels of VP1protein in BKPyV infected human epithelial cells. Western blot analysisof HK2 cellular lysates harvested 7 days post-infection with BKpolyomavirus at a multiplicity of infection of ~100. As compared toscramble AON-treated HK2 cells, AON #2, #3 and #4 clearly abrogateexpression levels of VP1 protein (n=4).

FIG. 6 : TAg splice-modulating AONs reduce BK virus replication. ViralDNA concentrations in the culture supernatant were determined by the PCRanalysis method for VP1 in the BKPyV genome. HK2 cells treated with AONs#2, #3 and #4 consistently reveal reduced levels of BKPyV genome in theculture supernatant, as compared to those treated with the scrambledAON.

FIG. 7 : Chemical modifications to AONs. Depicted are some chemicalmodifications that have been applied to the current TAgsplice-modulating AONs (phosphorothioate backbone and 2′-OMe on ribosemoiety). Other embodiments of these AONs could employ 2′-MOE or 2′-cEtmodifications at this position. These modifications primarily serve toimprove AON stability.

FIG. 8 : Sequence similarity for the 13 polyomaviruses known to havehuman hosts. Of note, these strains cover the alpha, beta and deltagenus, the majority of which have recently been identified. As shown inthe phylogenetic tree information on the left side of the figure, BKPyVand JCPyV share considerable sequence similarity, suggesting that theirco-localization in the proximal tubule epithelial cells of the kidneylends them to targeting with the herein described splice-modulatingAONs.

FIG. 9 : Design of AONs that could be employed to target otherpolyomaviruses. Alongside the aforementioned BK and JC virus TAgsplice-modulating AONs, AONs have been designed based on the possibilitythat they could also target the human polyomavirus 3 (KarolinskaInstitute or KI), human polyomavirus 4 (Washington University or WU) andhuman polyomavirus 5 (Merkel Cell virus or MCV). For humanpolyomaviruses 3-5, 2 AONs have been designed targeting the exon 1 –intron junction, and 2 for the intron – exon 2 junction, as opposed to 3AONs at the exon 1 – intron site.

FIG. 10 : Schematic of BK viral genome and BKV-targeting AONs. Part 1:The BK virus genome encodes six primary proteins, namely small t andlarge T antigen (early genes), as well as agnoprotein and the majorcapsid proteins VP1, VP2 and VP3 (late genes). The viral genome alsocontains a non-coding region (NCCR) whose sequence possesses both anorigin of replication, as well as the promoter region that isresponsible for recruiting transcription factors that drive expressionearly and late gene expression, while also co-ordinating viral genomereplication. As shown in FIG. 1 , alternative splicing of early and latepre-mRNAs results in numerous protein isoforms, including small tantigen, truncated T antigen (2 introns) and large T antigen (1 intron),along with alternative splicing determining the proportion of VP1, VP2or VP3 that is generated. The primary splice variant generated for thelate region predominantly results in expression of VP1. Part 2: AONsequences used to target the exon-intron junction of BKV large T antigen(TAg) as depicted in top panel. Part 3: Schematic depicting bindingsites for AONs at the exon - intron junction of BKV TAg.

FIG. 11 : Bioinformatic analysis of TAg splice site conservation fordesign of universal BKV-targeting AONs. Part 1: Phylogenetic treecontaining whole gene TAg sequences for unique BKV isolates/strainsshowing clear distinctions between BKV subgroups. Part 2: Sequence logosfor TAg splice sites with flanking regions (20 nucleotides) showing ahigh sequence conservation between subgroups. Sequences of antisenseoligonucleotides (AONs) directed towards the exon 1 – intron junction(HYB_01, HYB_02, HYB_03, HYB_06, HYB_07, HYB_08, HYB_09, HYB_10 andHYB_11) and intron - exon 2 junction (HYB_04, HYB_05, HYB_12, andHYB_13) of BKV TAg are provided.

FIG. 12 : TAg splice-modulating AONs reduce expression levels of TAgmRNAs in BKV-infected human kidney epithelial cells. Reduction in TAgRNA levels in scramble AON-treated HK2 cells versus HK2 cells treatedwith BKV-targeting AONs, after which cells were infected with BKV at amultiplicity of infection of ~100 (n=3 biological replicates). Note:HYB_14 binds exclusively to the exonic region of TAg exon 1 and does nottarget an exon - intron boundary.

FIG. 13 : TAg splice-modulating AONs reduce expression levels of VP1mRNAs in BKV-infected human kidney epithelial cells. Reduction in VP1RNA levels in scramble AON-treated HK2 cells versus HK2 cells treatedwith BKV-targeting AONs, after which cells were infected with BKV at amultiplicity of infection of ~100 (n=3 biological replicates). Note:HYB_14 binds exclusively to the exonic region of TAg exon 1 and does nottarget an exon - intron boundary.

FIG. 14 : TAg splice-modulating AONs reduce VP1 protein expressionlevels in BKV-infected human kidney epithelial cells. RepresentativeWestern blot analysis of cellular lysates harvested from BKV-targetingAON-treated HK2 cells. Blot depicts VP1 protein levels of lysatesharvested 7 days post-infection with BK polyomavirus at a multiplicityof infection of ~100 (n=3 biological replicates).

FIG. 15 : Quantification of VP1 protein expression levels in humankidney epithelial cells following treatment with BKV-targeting AONs andinfected with BKV. Quantification of Western blot analysis of HK2cellular lysates harvested 7 days post-infection with BK polyomavirus ata multiplicity of infection of ~100. Scramble AON-treated HK2 cells wereused as a control, and all values are in log₂ scale (n=3 biologicalreplicates). Note: HYB_14 binds exclusively to the exonic region of TAgexon 1 and does not target an exon - intron boundary.

FIG. 16 : TAg splice-modulating AONs reduce BKV DNA replication. Viralparticle concentrations in the culture supernatant at 7 dayspost-infection were determined by the PCR analysis method for VP1 in theBKV genome. HK2 cells treated with BKV-targeting AONs consistentlyreveal reduced levels of BKV particles in the culture supernatant, ascompared to those treated with the scrambled AON. Data represent abiological n=3.

FIG. 17 : TAg splice-modulating AONs reduce levels of HK2 re-infection.Culture supernatant was removed at 7 days from HK2 cells that had beenpre-treated with BKV-targeting AONs and infected with BKV. Thesupernatant was used to infect untreated HK2 cells (2h) after which thecells were cultured for 7 days and stained immunohistochemically for TAgand hoechst (for nuclei). Subsequently, the percent positive cells weredetermined and depicted relative to scramble AON-treated cells. Data arerepresentative of biological n=3.

FIG. 18 : Heatmap depicting cumulative effects of BKV-targeting AONtreatment on various aspects of BKV infection of HK2 cells. Summary ofeffects observed on TAg and VP1 mRNA, VP1 protein, viral particleproduction and re-infection. Scale indicates that black representslittle-to-no effect while white indicates large effect (2log fold changecompared to scrambled, n=3).

FIG. 19 : TAg splice-modulating lead AONs reduce expression levels ofTAg mRNAs in BKV-infected human kidney epithelial cells. Reduction inTAg RNA levels in scramble AON-treated HK2 cells versus HK2 cellstreated with BKV-targeting lead AONs (HYB_01, HYB_03 or HYB_11), afterwhich cells were infected with BKV at a multiplicity of infection of~100 (n=3 biological replicates). Note: SAN_73 and SAN_74 are previouslydescribed AONs (16 nucleotides in length). These data are representativeof a biological n=3.

FIG. 20 : TAg splice-modulating lead AONs reduce expression levels ofVP1 mRNAs in BKV-infected human kidney epithelial cells. Reduction inVP1 mRNA levels in scramble AON-treated HK2 cells versus HK2 cellstreated with BKV-targeting lead AONs (HYB_01, HYB_03 or HYB_11), afterwhich cells were infected with BKV at a multiplicity of infection of~100. Note: SAN_73 and SAN_74 are previously described AONs (16nucleotides in length). These data are representative of a biologicaln=3.

FIG. 21 : TAg splice-modulating lead AONs reduce VP1 protein expressionlevels in BKV-infected human kidney epithelial cells. RepresentativeWestern blot visualization of VP1 protein levels in cellular lysatesharvested from HK2 cells treated with BKV-targeting lead compound AONsand 7 days post-infection with BK polyomavirus at a multiplicity ofinfection of ~100 (n=3 biological replicates).

FIG. 22 : TAg splice-modulating lead AONs reduce expression levels ofVP1 protein in BKV-infected human kidney epithelial cells. Reduction inVP1 protein levels in scramble AON-treated HK2 cells versus HK2 cellstreated with BKV-targeting lead AONs (HYB_01, HYB_03 or HYB_11), afterwhich cells were infected with BKV at a multiplicity of infection of~100. Note: SAN_73 and SAN_74 are previously described AONs (16nucleotides in length). These data are representative of a biologicaln=3.

FIG. 23 : TAg splice-modulating lead AONs reduce viral particleproduction in BKV-infected human kidney epithelial cells. Reduction inthe levels of viral particles in scramble AON-treated HK2 cells versusHK2 cells treated with BKV-targeting lead AONs (HYB_01, HYB_03 orHYB_11), after which cells were infected with BKV at a multiplicity ofinfection of ~100. Note: SAN_73 and SAN_74 are previously described AONs(16 nucleotides in length). These data are representative of abiological n=3.

FIG. 24 : TAg splice-modulating lead AONs reduce levels of HK2re-infection. Culture supernatant was removed at 7 days from HK2 cellsthat had been pre-treated with BKV-targeting lead AONs (HYB_01, HYB_03or HYB_11) and infected with BKV. The supernatant was used to infectuntreated HK2 cells (2h) after which the cells were cultured for 7 daysand stained immunohistochemically for TAg and Hoechst (for nuclei).Subsequently, the percent positive cells were determined and depictedrelative to scramble AON-treated cells. Data are representative ofbiological n=3.

FIG. 25 : Heatmap depicting cumulative effects of BKV-targeting leadcompound AON treatment on various aspects of BKV infection of HK2 cells.Summary of effects observed on TAg and VP1 mRNA, VP1 protein, viralparticle production and re-infection for HK2 cells pre-treated with ourlead compound AONs (HYB_01, HYB_03 or HYB_11). Scale indicates thatblack represents little-to-no effect while white indicates large effect(2log fold change compared to scrambled, n=3).

FIG. 26 : Efficacy of TAg splice-modulating AON to reduce TAg mRNAexpression in HK2 pre-infected with BKV. As opposed to pre-AONtreatment, we first infected HK2 cells with BKV, and subsequentlyassessed the efficacy with which the AONs could reduce TAg mRNAexpression levels. Left panel: single dosing of AONs post-infectionsignificantly reduced expression levels of TAg mRNA, albeit thataddition of AON at later timepoints appears less efficacious. Rightpanel: multiple doses of the BKV-targeting AON more potently reduces TAgmRNA expression levels. It is noted that RNA expression levels for alltime points of treatment were determined at t = 7 post infection,resulting in shorter exposures to treatment for later time pointscompared to early treatment. Data are representative of biological n=1.

FIG. 27 : Efficacy of TAg splice-modulating AON to reduce VP1 mRNAexpression in HK2 pre-infected with BKV. As opposed to pre-AONtreatment, we first infected HK2 cells with BKV, and subsequentlyassessed the efficacy with which the AONs could reduce VP1 mRNAexpression levels. Left panel: single dosing of AONs post-infectionsignificantly reduced expression levels of VP1 mRNA, albeit thataddition of AON at later timepoints appears less efficacious. Rightpanel: multiple doses of the BKV-targeting AON more potently reduces VP1mRNA expression levels. It is noted that RNA expression levels for alltime points of treatment were determined at t = 7 post infection,resulting in shorter exposures to treatment for later time pointscompared to early treatment. Data are representative of biological n=1.

FIG. 28 : TAg splice-modulating lead AONs reduce expression levels ofTAg mRNAs in BKV-infected human primary proximal tubule epithelial cells(hPTECs). Reduction in TAg RNA levels in scramble AON-treated hPTECsversus hPTECs treated with BKV-targeting lead AONs (HYB_01, HYB_03 orHYB_11) or HYB_14, after which cells were infected with BKV at amultiplicity of infection of ~100 (n=3 biological replicates). Thesedata are representative of a biological n=3.

FIG. 29 : TAg splice-modulating lead AONs reduce expression levels ofVP1 mRNAs in BKV-infected hPTECs. Reduction in VP1 RNA levels inscramble AON-treated hPTECs versus hPTECs treated with BKV-targetinglead AONs (HYB_01, HYB_03 or HYB_11) or HYB_14, after which cells wereinfected with BKV at a multiplicity of infection of ~100 (n=3 biologicalreplicates). These data are representative of a biological n=3.

FIG. 30 : TAg splice-modulating lead AONs reduce VP1 and VP3 proteinexpression levels in BKV-infected hPTECs. Representative Western blotvisualization of VP1 and VP3 protein levels in cellular lysatesharvested from hPTECs treated with BKV-targeting lead compound AONs and7 days post-infection with BK polyomavirus at a multiplicity ofinfection of ~100 (VP1 and GAPDH: n=3 biological replicates, VP3: n=1).

FIG. 31 : TAg splice-modulating lead AONs reduce expression levels ofVP1 proteins in BKV-infected hPTECs. Reduction in VP1 protein levels inscramble AON-treated hPTECs versus hPTECs treated with BKV-targetinglead AONs (HYB_01, HYB_03 or HYB_11) or HYB_14, after which cells wereinfected with BKV at a multiplicity of infection of ~100 (n=3 biologicalreplicates). These data are representative of a biological n=3.

FIG. 32 : TAg splice-modulating lead AONs reduce expression levels ofVP3 proteins in BKV-infected hPTECs. Reduction in VP3 protein levels inscramble AON-treated hPTECs versus hPTECs treated with BKV-targetinglead AONs (HYB_01, HYB_03 or HYB_11) or HYB_14, after which cells wereinfected with BKV at a multiplicity of infection of ~100 (n=3 biologicalreplicates). These data are representative of a biological n=1.

FIG. 33 : TAg splice-modulating lead AONs reduce viral particleproduction in BKV-infected hPTECs. Reduction in the levels of viralparticles in scramble AON-treated hPTECs versus hPTECs treated withBKV-targeting lead AONs (HYB_01, HYB_03 or HYB_11) or HYB_14, afterwhich cells were infected with BKV at a multiplicity of infection of~100. These data are representative of a biological n=3.

FIG. 34 : Heatmap depicting cumulative effects of BKV-targeting leadcompound AON treatment on various aspects of BKV infection of hPTECs.Summary of effects observed on TAg and VP1 mRNA, VP1 protein and viralparticle production for hPTECs pre-treated with our lead compound AONs(HYB_01, HYB_03 or HYB_11) or HYB_14. Scale indicates that blackrepresents little-to-no effect while white indicates large effect (2logfold change compared to scrambled, n=3).

FIG. 35 : RNA-seq analysis of modulation of splicing induced by leadBKV-targeting AONs. Primary transcripts and splicing events are depictedin upper portion of panel, while in lower portion of panel the observedsplicing patterns and z-scores are presented, indicating that ourBKV-targeting AONs indeed modulate splicing. Events are observed at theexon 1 - intronic site as well as at the 3′-region of exon 2.Collectively, the presence of a single AON that appears to impact bothproximal and distal effects in splicing is indicative of a complexsplicing event.

FIG. 36 : Long-sequence read analysis of splicing modulation induced byBKV-targeting lead compound AONs. Long-sequence read analysis of RNAtranscripts from HK2 cells pre-treated with scrambled AON or our leadcompound AONs (HYB_01, HYB_03 or HYB_11). Left: Bar graphs depict thepercentage of transcripts utilizing the Large T or small t donor site incombination with a fixed acceptor site (intron - exon 2) or unsplicedtranscript, indicative of splicing modulation by BKV-targeting AONs.Right: Bar graphs depict the percentage of transcripts utilizing thetruncated T or Truncated T * acceptor site in combination with a fixeddonor site, indicative of splicing modulation by BKV-targeting AONs.

FIG. 37 : Schematic of JC viral genome and JCV-targeting AONs. Part 1:Similar to BK virus, the JC virus genome encodes small t and large Tantigen (early genes), as well as agnoprotein and the major capsidproteins VP1, VP2 and VP3 (late genes). The viral genome also contains anon-coding region (NCCR) with an origin of replication and promoterregion for transcription factor binding that drives expression of theearly and late genes, and viral genome replication. In contrast toextensive knowledge regarding BKV splicing of early and late regionpre-mRNAs, less is known regarding JCV splicing. Part 2: AON sequencesused to target the exon-intron junction of JCV large T antigen (TAg) asdepicted in top panel. Part 3: Schematic depicting binding sites forAONs at the exon - intron junction of JCV TAg.

FIG. 38 : TAg splice-modulating AONs reduce expression levels of TAgmRNAs in JCV-infected astrocytes derived human induced pluripotent stemcells. Reduction in TAg mRNA levels in scramble AON-treatedhiPSC-derived astrocytes versus hiPSC-derived astrocytes treated withJCV-targeting AONs, after which cells were infected with JCV (n=1).

FIG. 39 : TAg splice-modulating AONs reduce expression levels of VP1mRNAs in JCV-infected astrocytes derived human induced pluripotent stemcells. Reduction in VP1 mRNA levels in scramble AON-treatedhiPSC-derived astrocytes versus hiPSC-derived astrocytes treated withJCV-targeting AONs, after which cells were infected with JCV (n=1).

FIG. 40 : TAg splice-modulating AONs reduce expression levels of TAgmRNAs in JCV-infected primary human astrocytes. Reduction in TAg mRNAlevels in scramble AON-treated primary astrocytes versus primaryastrocytes treated with JCV-targeting AONs, after which cells wereinfected with JCV (n=2 biological replicates).

FIG. 41 : TAg splice-modulating AONs reduce expression levels of VP1mRNAs in JCV-infected primary human astrocytes. Reduction in VP1 mRNAlevels in scramble AON-treated primary astrocytes versus those treatedwith JCV-targeting AONs, after which cells were infected with JCV (n=2biological replicates).

FIG. 42 : Coverage of BKV genome by RNAs amplified during RNA-seq.Alignment of paired-end reads to the BKV genome from scrambled control,HYB_01, HYB_03, HYB_11, HYB_14 or SAN_73 and SAN_74 treated cells allowsfor semi-quantitative assessment of BKV RNA expression levels. Data areindicative of a biological n=3 and have been separated into early andlate phase gene expression profiles.

FIG. 43 : Electrophoretic analysis of long-read high-fidelity Phusionpolymerase generated TAg (pre-)mRNAs. Phusion polymerase was used togenerate long-read high fidelity mRNAs from RNA harvested from HK2 cellstreated with either scramble control AON (lane 1) or BKV-targeting AONs(namely HYB_01, HYB_03 or HYB_11; lanes 2-4, respectively) and assessedby capillary electrophoresis. Data are representative of a biologicaln=3.

FIG. 44 : Fluorescence microscopy of AON uptake in mouse kidneys 24hours after intravenous administration. Color separatedhigh-magnification images of mouse kidney sections 24 hours afterintravenous administration of 40 mg/kg HYB_01 (2′MOE without 5′ 6-FAMlabel) in C57BL/6J mice. Nuclei are stained with Hoechst, proximaltubule epithelial cell uptake is evident based on co-localization withlotus tetragonolobus lectin (LTL)-positive cells of the kidney (proximaltubuli). Left panels represent a 100x magnification (10x objective,scale bar = 100 µm) whereas right panels are 400x magnified (40xobjective, scale bar = 20 µm).

FIG. 45 : Immunohistochemical staining of mouse tissues forBKV-targeting AON uptake. Organs were excised 24 h after intravenousadministration of 40 mg/kg HYB_01 (2′MOE without 5′ 6-FAM label) inC57BL/6J mice and AON uptake assessed immunohistochemically. Hematoxylinand eosin staining (H&E) preceded the specific detection of the AONbackbone with anti-phosphorothioate antibody and diaminobenzidine (DAB)as peroxidase substrate to reveal the HRP-labelled secondary antibody.Positive signal for AON staining was visualized by color deconvolutionand thresholding in ImageJ, indicating positive tubuli with high levelsof AON uptake, with markedly reduced signal in liver and absence thereofin heart tissue.

EXAMPLES Example 1 Material and Methods Accessions Used for PhylogeneticAnalysis

Complete genomic sequences of BK polyomavirus isolates were downloadedfrom the publicly available NCBI database. From these records, only theisolates reporting a complete genome were used for the conservation ofthe splice sites in TAg. The Dunlop strain was used as a referencegenome. Isolates “MM” and “FNL-9” were removed due to a large deletionin the intron or duplication overlapping the acceptor splice siterespectively. Accession numbers of the 245 unique genomic sequences areprovided below:

AB211369.1; AB211370.1; AB211371.1; AB211372.1; AB211373.1; AB211374.1;AB211375.1; AB211376.1; AB211377.1; AB211378.1; AB211379.1; AB211381.1;AB211382.1; AB211383.1; AB211384.1; AB211385.1; AB211386.1; AB211387.1;AB211388.1; AB211389.1; AB211390.1; AB211391.1; AB213487.1; AB217917.1;AB217918.1; AB217919.1; AB217920.1; AB217921.1; AB260028.1; AB260029.1;AB260030.1; AB260031.1; AB260032.1; AB260033.1; AB263912.1; AB263913.1;AB263914.1; AB263915.1; AB263916.1; AB263917.1; AB263918.1; AB263919.1;AB263920.1; AB263921.1; AB263922.1; AB263923.1; AB263924.1; AB263925.1;AB263926.1; AB263927.1; AB263928.1; AB263929.1; AB263930.1; AB263931.1;AB263932.1; AB263934.1; AB263935.1; AB263936.1; AB263938.1; AB269825.1;AB269826.1; AB269827.1; AB269828.1; AB269829.1; AB269830.1; AB269831.1;AB269832.1; AB269834.1; AB269836.1; AB269837.1; AB269838.1; AB269840.1;AB269841.1; AB269842.1; AB269843.1; AB269844.1; AB269845.1; AB269846.1;AB269847.1; AB269848.1; AB269849.1; AB269850.1; AB269851.1; AB269852.1;AB269853.1; AB269854.1; AB269855.1; AB269856.1; AB269857.1; AB269858.1;AB269859.1; AB269860.1; AB269861.1; AB269862.1; AB269863.1; AB269864.1;AB269865.1; AB269866.1; AB269867.1; AB269868.1; AB269869.1; AB298941.1;AB298942.1; AB298945.1; AB298946.1; AB298947.1; AB301086.1; AB301087.1;AB301089.1; AB301090.1; AB301091.1; AB301092.1; AB301093.1; AB301094.1;AB301095.1; AB301096.1; AB301097.1; AB301099.1; AB301100.1; AB301101.1;AB365130.1; AB365132.1; AB365133.1; AB365134.1; AB365136.1; AB365137.1;AB365138.1; AB365139.1; AB365140.1; AB365141.1; AB365142.1; AB365144.1;AB365145.1; AB365146.1; AB365148.1; AB365149.1; AB365150.1; AB365151.1;AB365153.1; AB365154.1; AB365156.1; AB365157.1; AB365158.1; AB365159.1;AB365160.1; AB365162.1; AB365164.1; AB365165.1; AB365166.1; AB365167.1;AB365168.1; AB365170.1; AB365173.1; AB365174.1; AB365175.1; AB365176.1;AB365178.1; AB369087.1; AB369088.1; AB369089.1; AB369090.1; AB369092.1;AB369093.1; AB369094.1; AB369095.1; AB369096.1; AB369097.1; AB369098.1;AB369099.1; AB369101.1; AB464953.1; AB464954.1; AB464956.1; AB464957.1;AB464958.1; AB464960.1; AB464961.1; AB464962.1; AB485695.1; AB485696.1;AB485697.1; AB485698.1; AB485699.1; AB485700.1; AB485701.1; AB485703.1;AB485704.1; AB485707.1; AB485709.1; AB485710.1; AB485711.1; AB485712.1;AY628224.1; AY628225.1; AY628226.1; AY628227.1; AY628228.1; AY628229.1;AY628230.1; AY628231.1; AY628232.1; AY628233.1; AY628234.1; AY628235.1;AY628236.1; AY628237.1; AY628238.1; DQ305492.1; EF376992.1; FR720308.1;FR720309.1; FR720310.1; FR720311.1; FR720312.1; FR720313.1; FR720315.1;FR720317.1; FR720318.1; FR720320.1; FR720321.1; JF894228.1; JN192431.1;JN192432.1; JN192433.1; JN192435.1; JN192437.1; JN192438.1; JN192439.1;JN192440.1; JQ713822.1; KF055891.1; KF055892.1; KF055893.1; KP412983.1;KP984526.1; KY114802.1; KY114803.1; KY132094.1; KY487998.1; LC029413.1;LC309239.1; LC309240.1; LT960370.1; M23122.1; V01108.1.

Similarly, complete genomic sequences were downloaded for the 13different prototype human polyomaviruses. The accession numbers aredepicted below:

NC_001538; NC_001699; NC_009238; NC_009539; NC_010277; NC_014406;NC_014407; NC_014361; NC_015150; NC_018102; NC_020106; NC_020890;NC_024118.

Conservation of Large T Antigen Splice Sites

Whole genome nucleotide sequences from all reference humanpolyomaviruses were downloaded from the NCBI websitehttps://www.ncbi.nlm.nih.gov/nucore) on Feb. 20, 2018 and aligned withWebPrank (available online: https://www.ebi.ac.uk/goldman-srv/webprank/)using default settings. A phylogenetic UPGMA tree was constructed andsequence logos for every splice site were created to show conservationbetween different human polyomaviruses. All downloaded refseq accessionnumbers are depicted below.

Reference sequences:

NC_001538, NC_001699, NC_009238, NC_009539, NC_010277, NC_014406,NC_014407, NC_014361, NC_015150, NC_018102, NC_020106, NC_020890,NC_024118

Whole genome nucleotide sequences for all human polyomavirus isolateswere downloaded from the NCBI website on Feb. 20, 2018. Whole genesequences of Large T antigen were retrieved from only the unique genomicsequences and aligned with WebPrank using default settings Sequencelogos were created for every splice site in Large T antigen to showconservation within and between different human polyomaviruses.

All downloaded accession numbers are depicted below:

BKPyV: AB211369.1, AB211370.1, AB211371.1, AB211372.1, AB211373.1,AB211374.1, AB211375.1, AB211376.1, AB211377.1, AB211378.1, AB211379.1,AB211380.1, AB211381.1, AB211382.1, AB211383.1, AB211384.1, AB211385.1,AB211386.1, AB211387.1, AB211388.1, AB211389.1, AB211390.1, AB211391.1,AB213487.1, AB217917.1, AB217918.1, AB217919.1, AB217920.1, AB217921.1,AB260028.1, AB260029.1, AB260030.1, AB260031.1, AB260032.1, AB260033.1,AB260034.1, AB263912.1, AB263913.1, AB263914.1, AB263915.1, AB263916.1,AB263917.1, AB263918.1, AB263919.1, AB263920.1, AB263921.1, AB263922.1,AB263923.1, AB263924.1, AB263925.1, AB263926.1, AB263927.1, AB263928.1,AB263929.1, AB263930.1, AB263931.1, AB263932.1, AB263933.1, AB263934.1,AB263935.1, AB263936.1, AB263937.1, AB263938.1, AB269822.1, AB269823.1,AB269824.1, AB269825.1, AB269826.1, AB269827.1, AB269828.1, AB269829.1,AB269830.1, AB269831.1, AB269832.1, AB269833.1, AB269834.1, AB269835.1,AB269836.1, AB269837.1, AB269838.1, AB269839.1, AB269840.1, AB269841.1,AB269842.1, AB269843.1, AB269844.1, AB269845.1, AB269846.1, AB269847.1,AB269848.1, AB269849.1, AB269850.1, AB269851.1, AB269852.1, AB269853.1,AB269854.1, AB269855.1, AB269856.1, AB269857.1, AB269858.1, AB269859.1,AB269860.1, AB269861.1, AB269862.1, AB269863.1, AB269864.1, AB269865.1,AB269866.1, AB269867.1, AB269868.1, AB269869.1, AB298940.1, AB298941.1,AB298942.1, AB298943.1, AB298944.1, AB298945.1, AB298946.1, AB298947.1,AB301086.1, AB301087.1, AB301088.1, AB301089.1, AB301090.1, AB301091.1,AB301092.1, AB301093.1, AB301094.1, AB301095.1, AB301096.1, AB301097.1,AB301098.1, AB301099.1, AB301100.1, AB301101.1, AB301102.1, AB301103.1,AB365130.1, AB365131.1, AB365132.1, AB365133.1, AB365134.1, AB365135.1,AB365136.1, AB365137.1, AB365138.1, AB365139.1, AB365140.1, AB365141.1,AB365142.1, AB365143.1, AB365144.1, AB365145.1, AB365146.1, AB365147.1,AB365148.1, AB365149.1, AB365150.1, AB365151.1, AB365152.1, AB365153.1,AB365154.1, AB365155.1, AB365156.1, AB365157.1, AB365158.1, AB365159.1,AB365160.1, AB365161.1, AB365162.1, AB365163.1, AB365164.1, AB365165.1,AB365166.1, AB365167.1, AB365168.1, AB365169.1, AB365170.1, AB365171.1,AB365172.1, AB365173.1, AB365174.1, AB365175.1, AB365176.1, AB365177.1,AB365178.1, AB369087.1, AB369088.1, AB369089.1, AB369090.1, AB369091.1,AB369092.1, AB369093.1, AB369094.1, AB369095.1, AB369096.1, AB369097.1,AB369098.1, AB369099.1, AB369100.1, AB369101.1, AB464953.1, AB464954.1,AB464955.1, AB464956.1, AB464957.1, AB464958.1, AB464959.1, AB464960.1,AB464961.1, AB464962.1, AB464963.1, AB485694.1, AB485695.1, AB485696.1,AB485697.1, AB485698.1, AB485699.1, AB485700.1, AB485701.1, AB485702.1,AB485703.1, AB485704.1, AB485705.1, AB485706.1, AB485707.1, AB485708.1,AB485709.1, AB485710.1, AB485711.1, AB485712.1, AY628224.1, AY628225.1,AY628226.1, AY628227.1, AY628228.1, AY628229.1, AY628230.1, AY628231.1,AY628232.1, AY628233.1, AY628234.1, AY628235.1, AY628236.1, AY628237.1,AY628238.1, DQ305492.1, EF376992.1, FR720308.1, FR720309.1, FR720310.1,FR720311.1, FR720312.1, FR720313.1, FR720314.1, FR720315.1, FR720316.1,FR720317.1, FR720318.1, FR720319.1, FR720320.1, FR720321.1, FR720322.1,FR720323.1, JF894228.1, JN192431.1, JN192432.1, JN192433.1, JN192434.1,JN192435.1, JN192436.1, JN192437.1, JN192438.1, JN192439.1, JN192440.1,JN192441.1, JQ713822.1, KF055891.1, KF055892.1, KF055893.1, KP412983.1,KP984526.1, KY114802.1, KY114803.1, KY132094.1, KY487998.1, LC029411.1,LC029412.1, LC029413.1, LC029414.1, LC309239.1, LC309240.1, LT934539.1,LT960370.1, M23122.1, MF627830.1, MF627831.1, V01108.1, V01109.1

JCPyV: AB038249.1, AB038250.1, AB038251.1, AB038252.1, AB038253.1,AB038254.1, AB038255.1, AB048545.1, AB048546.1, AB048547.1, AB048548.1,AB048549.1, AB048550.1, AB048551.1, AB048552.1, AB048553.1, AB048554.1,AB048555.1, AB048556.1, AB048557.1, AB048558.1, AB048559.1, AB048560.1,AB048561.1, AB048562.1, AB048563.1, AB048564.1, AB048565.1, AB048566.1,AB048567.1, AB048568.1, AB048569.1, AB048570.1, AB048571.1, AB048572.1,AB048573.1, AB048574.1, AB048575.1, AB048576.1, AB048577.1, AB048578.1,AB048579.1, AB048580.1, AB048581.1, AB048582.1, AB074575.1, AB074576.1,AB074577.1, AB074578.1, AB074579.1, AB074580.1, AB074581.1, AB074582.1,AB074583.1, AB074584.1, AB074585.1, AB074586.1, AB074587.1, AB074588.1,AB074589.1, AB074590.1, AB074591.1, AB077855.1, AB077856.1, AB077857.1,AB077858.1, AB077859.1, AB077860.1, AB077861.1, AB077862.1, AB077863.1,AB077864.1, AB077865.1, AB077866.1, AB077867.1, AB077868.1, AB077869.1,AB077870.1, AB077871.1, AB077872.1, AB077873.1, AB077874.1, AB077875.1,AB077876.1, AB077877.1, AB077878.1, AB077879.1, AB081005.1, AB081006.1,AB081007.1, AB081008.1, AB081009.1, AB081010.1, AB081011.1, AB081012.1,AB081013.1, AB081014.1, AB081015.1, AB081016.1, AB081017.1, AB081018.1,AB081019.1, AB081020.1, AB081021.1, AB081022.1, AB081023.1, AB081024.1,AB081025.1, AB081026.1, AB081027.1, AB081028.1, AB081029.1, AB081030.1,AB081600.1, AB081601.1, AB081602.1, AB081603.1, AB081604.1, AB081605.1,AB081606.1, AB081607.1, AB081608.1, AB081609.1, AB081610.1, AB081611.1,AB081612.1, AB081613.1, AB081614.1, AB081615.1, AB081616.1, AB081617.1,AB081618.1, AB081654.1, AB092578.1, AB092579.1, AB092580.1, AB092581.1,AB092582.1, AB092583.1, AB092584.1, AB092585.1, AB092586.1, AB092587.1,AB103387.1, AB103402.1, AB103403.1, AB103404.1, AB103405.1, AB103406.1,AB103407.1, AB103408.1, AB103409.1, AB103410.1, AB103411.1, AB103412.1,AB103413.1, AB103414.1, AB103415.1, AB103416.1, AB103417.1, AB103418.1,AB103419.1, AB103420.1, AB103421.1, AB103422.1, AB103423.1, AB104487.1,AB113118.1, AB113119.1, AB113120.1, AB113121.1, AB113122.1, AB113123.1,AB113124.1, AB113125.1, AB113126.1, AB113127.1, AB113128.1, AB113129.1,AB113130.1, AB113131.1, AB113132.1, AB113133.1, AB113134.1, AB113135.1,AB113136.1, AB113137.1, AB113138.1, AB113139.1, AB113140.1, AB113141.1,AB113142.1, AB113143.1, AB113144.1, AB113145.1, AB113216.1, AB113217.1,AB118231.1, AB118232.1, AB118233.1, AB118234.1, AB118235.1, AB118651.1,AB118652.1, AB118653.1, AB118654.1, AB118655.1, AB118656.1, AB118657.1,AB118658.1, AB118659.1, AB126981.1, AB126982.1, AB126983.1, AB126984.1,AB126985.1, AB126986.1, AB126987.1, AB126988.1, AB126989.1, AB126990.1,AB126991.1, AB126992.1, AB126993.1, AB126994.1, AB126995.1, AB126996.1,AB126997.1, AB126998.1, AB126999.1, AB127000.1, AB127001.1, AB127002.1,AB127003.1, AB127004.1, AB127005.1, AB127006.1, AB127007.1, AB127008.1,AB127009.1, AB127010.1, AB127011.1, AB127012.1, AB127013.1, AB127014.1,AB127015.1, AB127016.1, AB127017.1, AB127018.1, AB127019.1, AB127020.1,AB127021.1, AB127022.1, AB127023.1, AB127024.1, AB127025.1, AB127026.1,AB127027.1, AB127342.1, AB127343.2, AB127344.1, AB127345.2, AB127346.1,AB127347.1, AB127348.1, AB127349.1, AB127350.2, AB127351.2, AB127352.1,AB127353.1, AB183152.1, AB195639.1, AB195640.1, AB198940.1, AB198941.1,AB198942.1, AB198943.1, AB198944.1, AB198945.1, AB198946.1, AB198947.1,AB198948.1, AB198949.1, AB198950.1, AB198951.1, AB198952.1, AB198953.1,AB198954.1, AB220939.1, AB220940.1, AB220941.1, AB220942.1, AB220943.1,AB262396.1, AB262397.1, AB262398.1, AB262399.1, AB262400.1, AB262401.1,AB262402.1, AB262403.1, AB262404.1, AB262405.1, AB262406.1, AB262407.1,AB262408.1, AB262409.1, AB262410.1, AB262411.1, AB262412.1, AB262413.1,AB362351.1, AB362352.1, AB362353.1, AB362354.1, AB362355.1, AB362356.1,AB362357.1, AB362358.1, AB362359.1, AB362360.1, AB362361.1, AB362362.1,AB362363.1, AB362364.1, AB362365.1, AB362366.1, AB372036.1, AB372037.1,AB372038.1, AF030085.1, AF295731.1, AF295732.1, AF300945.1, AF300946.1,AF300947.1, AF300948.1, AF300949.1, AF300950.1, AF300951.1, AF300952.1,AF300953.1, AF300954.1, AF300955.1, AF300956.1, AF300957.1, AF300958.1,AF300959.1, AF300960.1, AF300961.1, AF300962.1, AF300963.1, AF300964.1,AF300965.1, AF300966.1, AF300967.1, AF363830.1, AF363831.1, AF363832.1,AF363833.1, AF363834.1, AY121907.1, AY121908.1, AY121909.1, AY121910.1,AY121911.1, AY121912.1, AY121913.1, AY121914.1, AY121915.1, AY328376.1,AY342299.1, AY349147.1, AY356539.1, AY364314.1, AY366359.1, AY373463.1,AY376828.1, AY376829.1, AY376830.1, AY376831.1, AY378084.1, AY378085.1,AY378086.1, AY378087.1, AY382184.1, AY382185.1, AY382186.1, AY382187.1,AY382188.1, AY386373.1, AY386374.1, AY386375.1, AY386376.1, AY386377.1,AY386378.1, AY536239.1, AY536240.1, AY536241.1, AY536242.1, AY536243.1,DQ875211.1, DQ875212.1, EU835194.1, JF424834.1, JF424835.1, JF424836.1,JF424837.1, JF424838.1, JF424839.1, JF424840.1, JF424841.1, JF424842.1,JF424843.1, JF424844.1, JF424845.1, JF424846.1, JF424847.1, JF424848.1,JF424849.1, JF424850.1, JF424851.1, JF424852.1, JF424853.1, JF424854.1,JF424855.1, JF424856.1, JF424857.1, JF424858.1, JF424859.1, JF424860.1,JF424861.1, JF424862.1, JF424863.1, JF424864.1, JF424865.1, JF424866.1,JF424867.1, JF424868.1, JF424869.1, JF424870.1, JF424871.1, JF424872.1,JF424873.1, JF424874.1, JF424875.1, JF424876.1, JF424877.1, JF424878.1,JF424879.1, JF424880.1, JF424881.1, JF424882.1, JF424883.1, JF424884.1,JF424885.1, JF424886.1, JF424887.1, JF424888.1, JF424889.1, JF424890.1,JF424891.1, JF424892.1, JF424893.1, JF424894.1, JF424895.1, JF424896.1,JF424897.1, JF424898.1, JF424899.1, JF424900.1, JF424901.1, JF424902.1,JF424903.1, JF424904.1, JF424905.1, JF424906.1, JF424907.1, JF424908.1,JF424909.1, JF424910.1, JF424911.1, JF424912.1, JF424913.1, JF424914.1,JF424915.1, JF424916.1, JF424917.1, JF424918.1, JF424919.1, JF424920.1,JF424921.1, JF424922.1, JF424923.1, JF424924.1, JF424925.1, JF424926.1,JF424927.1, JF424928.1, JF424929.1, JF424930.1, JF424931.1, JF424932.1,JF424933.1, JF424934.1, JF424935.1, JF424936.1, JF424937.1, JF424938.1,JF424939.1, JF424940.1, JF424941.1, JF424942.1, JF424943.1, JF424944.1,JF424945.1, JF424946.1, JF424947.1, JF424948.1, JF424949.1, JF424950.1,JF424951.1, JF424952.1, JF424953.1, JF424954.1, JF424955.1, JF424956.1,JF424957.1, JF424958.1, JF424959.1, JF424960.1, JF424961.1, JF424962.1,JF425488.1, JF425489.1, JF425490.1, JF425491.1, JF425492.1, JF425493.1,JF425494.1, JF425495.1, JF425496.1, JF425497.1, JF425498.1, JF425499.1,JF425500.1, JF425501.1, JF425502.1, JF425503.1, JF425504.1, JF425551.1,JF425552.1, JF425553.1, JF425554.1, JF425555.1, JF425556.1, JQ237146.1,JQ823124.1, JX273163.1, KJ659286.1, KJ659287.1, KJ659288.1, KJ659289.1,KM225765.1, LC164349.1, LC164350.1, LC164351.1, LC164352.1, LC164353.1,LC164354.1, MF662180.1, MF662181.1, MF662182.1, MF662183.1, MF662184.1,MF662185.1, MF662186.1, MF662187.1, MF662188.1, MF662189.1, MF662190.1,MF662191.1, MF662192.1, MF662193.1, MF662194.1, MF662195.1, MF662196.1,MF662197.1, MF662198.1, MF662199.1, MF662200.1, MF662201.1, MF662202.1,MF662203.1, MF662204.1

KIPyV: EF520287.1, EF520288.1, EF520289.1, EU358766.1, EU358767.1,KC571691.1, KM085447.1, KU746835.1

WUPyV: EF444549.1, EF444550.1, EF444551.1, EF444552.1, EF444553.1,EF444554.1, EU296475.1, EU358768.1, EU358769.1, EU711054.1, EU711055.1,EU711056.1, EU711057.1, EU711058.1, FJ794068.1, FJ890981.1, FJ890982.1,GQ926975.1, GQ926976.1, GQ926977.1, GQ926978.1, GQ926979.1, GQ926980.1,GU296361.1, GU296362.1, GU296363.1, GU296364.1, GU296365.1, GU296366.1,GU296367.1, GU296368.1, GU296369.1, GU296370.1, GU296371.1, GU296372.1,GU296373.1, GU296374.1, GU296375.1, GU296376.1, GU296377.1, GU296378.1,GU296379.1, GU296380.1, GU296381.1, GU296382.1, GU296383.1, GU296384.1,GU296385.1, GU296386.1, GU296387.1, GU296388.1, GU296389.1, GU296390.1,GU296391.1, GU296392.1, GU296393.1, GU296394.1, GU296395.1, GU296396.1,GU296397.1, GU296398.1, GU296399.1, GU296400.1, GU296401.1, GU296402.1,GU296403.1, GU296404.1, GU296405.1, GU296406.1, GU296407.1, GU296408.1,HQ218321.1, KC571693.1, KC571694.1, KC571695.1, KC571696.1, KC571697.1,KC571698.1, KC571699.1, KJ643309.1, KJ725028.1, KM265136.1, KU049032.1,KU672381.1, KX650181.1, KX650182.1, KX650184.1, KX650185.1, KX650186.1,KX650187.1, KX650188.1, KX650189.1, KX650190.1, KX650191.1, KX650192.1,KX650193.1

MCPyV: EU375803.1, EU375804.1, FJ173815.1, FJ464337.1, HM011538.1,HM011539.1, HM011540.1, HM011541.1, HM011542.1, HM011543.1, HM011544.1,HM011545.1, HM011546.1, HM011547.1, HM011548.1, HM011549.1, HM011550.1,HM011551.1, HM011552.1, HM011553.1, HM011554.1, HM011555.1, HM011556.1,HM011557.1, HM355825.1, JF812999.1, JF813000.1, JF813001.1, JF813002.1,JF813003.1, JN383838.1, JN383839.1, JN383840.1, JN383841.1, JQ479315.1,JQ479316.1, JQ479317.1, JQ479318.1, JQ479319.1, JQ479320.1, JX045708.1,JX045709.1, KC202810.1, KC571692.1, KF266963.1, KF266964.1, KF266965.1,KX781279.1, KX827417.1, NC_010277.2

HPyV6: HM011558.1, HM011559.1, HM011560.1, HM011561.1, HM011562.1,HM011563.1, KM387421.1, KM655817.1, KR090570.1, KU596573.1, KX379630.1,KX379631.1, KX771234.1

HPyV7: HM011564.1, HM011565.1, HM011566.1, HM011567.1, HM011568.1,HM011569.1, KJ733012.1, KJ733013.1, KX771235.1

TSPyV: AB873001.1, JQ723730.1, KF444091.1, KF444092.1, KF444093.1,KF444094.1, KF444095.1, KF444096.1, KF444097.1, KF444098.1, KF444099.1,KF444100.1, KF444101.1, KM007161.1, KM655816.1, KU221329.1, KX249740.1,KX249741.1, KX249742.1, KX249743.1

HPyV9: HQ696595.1 , KC831440.1

MWPyV: JQ898291.1, JQ898292.1, KC549586.1, KC549587.1, KC549588.1,KC549589.1, KC549590.1, KC549591.1, KC549592.1, KC549593.1, KC549594.1,KC571700.1, KC571701.1, KC571702.1, KC571703.1, KC571704.1, KC571705.1,KC690147.1, KR338953.1

STLPyV: JX463183.1, JX463184.1, KF525270.1, KF530304.1, KF651951.1,KM893862.1, KR090571.1, NC_020106.1

HPyV12: JX308829.1, NC_020890.1

NJPyV: KF954417.1, NC_024118.1

Splice Site Conservation and Phylogenetic Trees

Whole-gene sequences of TAg, including intron sequences, were alignedusing clustalW (“msa” package in R) for the 13 different polyomavirusreference sequences and all unique BK-polyomavirus isolates. Aphylogenetic tree was constructed using the UPGMA method (“phangorn” and“ggtree” packages in R). A sequence logo was constructed for theacceptor and donor splice sites to show nucleotide specific conservationbetween subtypes (“msa” package in R).

AON Design

Antisense oligonucleotides (AONs) were designed to target the splicesites in TAg. Ribonucleic acids in the AONs contain 2′-OMemodifications. AONs are 20 nucleotides in length with a fullphosphorothioate backbone (*). For in vitro studies the AONs contain a5′-FAM label. Secondary structure and binding energy of the AONs werepredicted using RNA structure. All AON sequences are depicted below:

Name Sequence Target splice site in TAg SEQ ID NO ScrambledG*C*A*C*C*U*C*U*G*C*G*U*C*C*U*A*G*A*A*T Not applicable 36 1_1A*C*C*U*C*U*G*A*G*C*U*A*C*U*C*C*A*G*G*U Donor (exon 1) 1 1_2A*C*A*A*A*C*C*U*C*U*G*A*G*C*U*A*C*U*C*C Donor (exon 1) 2 1_3C*A*G*C*A*C*A*A*A*C*C*U*C*U*G*A*G*C*U*A Donor (exon 1) 3 2_1U*C*C*A*U*A*G*G*U*U*G*G*C*A*C*C*U*A*G*A Acceptor (exon 2) 4 2_2U*G*U*U*C*C*A*U*A*G*G*U*U*G*G*C*A*C*C*U Acceptor (exon 2) 5 *Indicates aphosphorothioate linkage.

Cell Culture

Immortalized proximal tubule kidney epithelial HK2 cells (ATCC®CRL-2190™) were obtained from ATCC and maintained at 37° C., 5% CO₂, inDulbecco’s Modified Eagle’s medium-F12, 1:1 mixture with 15 mM Hepes,2.5 mM L-glutamine (Lonza) and supplemented with Tri-iodo thyronine,epidermal growth factor (EGF), insulin-transferrin-selenium-ethanolamine(ITS-X), hydrocortison and 100 U/mL penicillin-streptomycin. BKpolyomavirus (ATCC® VR-837™) was obtained from ATCC and diluted incomplete HK2 culture media to reduce the infectious load. For treatmentexperiments, cells were seeded in 6-, or 12- wells plates (Corning) at adensity of 32,000 cells/cm² and grown overnight. AON treatment wasperformed by incubating the cells for 5h with lipofectamine 3000 (ThermoFisher) at an AON concentration of 50 nM, after which the lipofectaminewas washed off. Infections with BK polyomavirus were performed 24 hafter washing of the cells by incubating the cells with BKpolyomavirus-containing culture media for 2 h, after which the virus waswashed off. Supernatant was collected after washing and at 3, 5 and 7days after infection to determine the production of viral particlesusing PCR. A viral load sample was collected before infection todetermine the infectious load. RNA and protein was harvested at day 7 todetermine the expression of TAg and VP1.

Viral Load Determinations

In order to determine the viral load in the culture supernatant, 200 µLwas collected from every well for every time point. Pierce UniversalNuclease was added to every sample to degrade unpackaged DNA for 15minutes at RT and was then inactivated with 5 mM EDTA. Viral DNA wasisolated from the supernatant using the DNA mini kit (Qiagen) and theviral load was determined using Taqman PCR as described below(Wunderink, H.F., et. al., J. Clin. Virol., 2017).

To monitor the quality of DNA extraction and potential PCR inhibition,we added low concentrations of phocine herpesvirus to the lysis buffer.DNA was eluted in a final volume of 100 µL elution buffer, of which 10µL was used as input for real-time quantitative PCR (qPCR). Using theprimers 440BKVs 5′-GAAAAGGAGAGT-GTCCAGGG-3′ (SEQ ID NO: 37) and 441BKVas5′-GAACTTCTACTCCTCCTT-TTATTAGT-3′ (SEQ ID NO:38) and a Taqman probe576BKV-TQ-FAM FAM 5′-CCAAAAAGCCAAAGGAACCC-3′-BHQ1 (SEQ ID NO:39), a90-bp fragment within the BKPyV VP1 gene was amplified. The BKPyV qPCRand phocine herpesvirus PCR were duplexed for DNA quality and potentialPCR inhibition monitoring. Furthermore, the BKPyV qPCR was validated todetect BKPyV genotypes I-IV.

Quantitative PCR reactions were performed in a total volume of 50 µL,containing 25 µL HotStarTaq Master Mix (QIAGEN, Hilden, Germany), 0.5µmol/L of each primer, 0.35 µmol/L BKPyV probe, and 3.5 mmol/L MgC12.Reactions were performed using a CFX96 real-time detection system(Bio-Rad, Hercules, CA, USA) with the following cycle conditions: 15 minat 95° C. followed by 45 cycles of amplification (30 s at 95° C.; 30 sat 55° C.; 30 s at 72° C.). For quantification, a standard of aquantified BKPyV-positive urine sample was used. Analytical sensitivityof the BKPyV qPCR was ~10 copies/mL. On each plate, 3 negative controlswere included; these controls tested negative in all PCR assays. PCRresults with a cycle threshold ≥40 were considered negative.

Antibodies and Western Blot

Protein concentrations were determined using the BCA method. Sampleswere run on a 4-15% TGX gel and transferred to a nitrocellulose or PVDFmembrane. Antibodies used were: rabbit polyclonal anti-actin-HRP(loading control), rabbit polyclonal anti -SV40 VP1 (ab53977, Abcam),mouse monoclonal anti-SV40 T-antigen [PAb416] (ab16879, Abcam) and mousemonoclonal anti-SV40 T-Antigen (PAb108, Thermo Fisher). The primaryantibody was incubated overnight at 4° C. for TAg and VP1 and 30 minutesat RT for actin. Secondary antibodies used for TAg and VP1 were goatpolyclonal anti-mouse-HRP (P044701-2, Agilent) and goat polyclonalanti-rabbit-HRP (P044801-2, Agilent) respectively. The membranes wereincubated with SuperSignal™ West Femto Maximum Sensitivity Substrate(Thermo Fisher) and protein bands were visualized using the ChemiDoc MPImaging System (Bio Rad).

Real-Time qPCR

BK-infected HK2 cells were lysed in Trizol and RNA was isolated usingthe RNeasy kit (Qiagen). A DNAse I (Qiagen) treatment was added toremove excess DNA during the isolation and cDNA was synthesized usingPromega reverse transcriptase, DTT, dNTPs and random primers. Real timePCR was performed on a CFX384 Touch™ Real-Time PCR Detection System (BioRad) with SYBR™ Select Master Mix (Thermo Fisher) and the followingprimers:

Gene Forward SEQ ID NO Reverse SEQ ID NO GAPDH ACAACTTTGGTATCGTGGAAGG 40GCCATCACGCCACAGTTTC 41 TAg GAGGAGGATGTAAAGGTAGCTCA 42ACTGGCAAACATATCTTCATGGC 43 VP1 TGCAGGGTCACAAAAAGTGC 44AGCACTCCCTGCATTTCCAA 45

Results Design of BK-Targeting Antisense Oligonucleotides

Efficient antisense oligonucleotides (AONs) that target the BKPyV largeT antigen (TAg) must be specific for BKPyV in the sense that they arenot specific for host RNA species, while preferably also being asuniversal to other different BKPyV isolates and different polyomavirusesin general as possible. dsDNA viruses (in this cases polyomaviruses) ascompared to dsRNA/ssRNA viruses are characterized by less genetic drift.Nonetheless, there remain a large number of BKPyV genotypes andsubgenotypes that give rise to a large number of BKPyV serotypes (seephylogenetic tree in FIG. 2 ). Since TAg contains two exons, we firstidentified the genomic sequence at the exon-intron junctions in attemptsto identify AON candidates (See FIG. 2 ). To achieve this, we extractedthe available unique BKPyV TAg genomic sequences (n=245 accessionnumbers provided in Material and Methods section) from the NCBI databaseand aligned these using ClustalW. Regions that depicted that a highlevel of sequence similarity (or conservation) were targeted. Thesestudies revealed a high degree of conservation in exon 1 and flankingintronic sequence (see FIG. 2 ). Exon 2 also displayed a high level ofsequence conservation. Flanking intronic sequence was both Tnucleotide-rich and less conserved within 4 nucleotides from the exonboundary. AON were targeted to exon 1 of BKPyV TAg and bridge portionsof intronic sequence at exon 1. For exon 2 AON were targeted to 4-6nucleotides in the intronic region of exon 2 and the flanking exon 2sequence.

As shown in FIG. 3 , we elected to design 5 AONs targeting BKPyV TAg, 3of which target the exon 1 – intron portion (designated AON #1, #2 and#3), while 2 AONs target the exon 2 – intron portion (termed AON #4 and#5). The AONs progressively shift from primarily exonic to includingsignificant intron-binding sequence for exon 1 targeting AONs (AON 1-3).See Material and Methods section and FIG. 3 for exact sequences, as wellas the backbone and sugar moiety modifications.

This design allowed us to specifically target the TAg of BKPyV, whilealso being universal for distinct BKPyV genotypes in kidney transplantpatients.

AON-Mediated Reduction in BKPyV TAg RNA

We employed lipofectamine-based delivery of the AONs, which markedlyimproved AON uptake within 5 hours after transfection. Moreover, wetitrated AON dosage based on FAM label cellular intensity to be maximalat approximately 50 nM. Twenty-four hours (24h) after AONadministration, HK2 cells were infected with BKV for 2 hours, afterwhich the cells were washed and cultured for 3, 5 and 7 days. At thesepoints, RNA was harvested from the cells and qRT-PCR performed todetermine which AONs could affect TAg expression levels. HK2 cells thatwere not transfected with AONs (untreated) displayed similar expressionlevels of TAg as compared to scrambled-AON (Scr) treated cells (data notshown).

It is well established that a considerable proportion of AONs designedto modulate expression levels or splicing of a given target RNA areefficacious. Our studies using the AONs #2, #3, or #4 repeatedlydisplayed significant reductions in TAg RNA levels, generally revealing5- to 10-fold attenuation in the RNA levels of this viral DNA driver(see FIG. 4 , left panel).

BKPyV-infected cultures that were treated with the AONs #2, #3, #4 or #5repeated exhibited diminished TAg RNA expression levels. Thisestablishes the sites targeted by these AON as good target sites forreducing BK virus production by reducing TAg production. Of note, thisreduction is observed in the setting a high MOI, namely in the range of100.

AON-Mediated Reduction of VP1 RNA and Protein

In cells latently infected by polyomavirus, such as BKV-infectedproximal tubule cells of the kidney, low levels of TAg RNA and proteinexpression are maintained. In individuals with a compromised immunesystem, be it natural or induced by an immunosuppressive regimen,replication of virus and induction of TAg expression is observed(Hasegawa, M. et. al., Transplantation Proceedings, 2014; Nickeleit, V.et. al., JASN, 2018). Augmentation of TAg levels, along with theinteraction with accessory transcription factors to thenon-coding/promoter region of the BKV genome drives both BK genomereplication and expression of the (late region) major capsid proteins.Collectively, the TAg-mediated activation of viral DNA replication andencapsulation by the capsid proteins results in the generation ofinfectious viral particles that can be detected in both the urine(viruria) and in the serum (viremia)(Helle, F. et. al., Viruses, 2017).

To test whether AONs are effective in reducing BKV generation wedetermined the expression profile of TAg-activated proteins, includingVP1. VP1 is the major structural constituent of the icosahedral viralcapsid. This outer shell has 72 pentamers that are joined in astoichiometry of 5:1 by either VP2 or VP3. As such, we performed qRT-PCRfor VP1, which revealed that expression levels of VP1 are much higherthan TAg per copies of GAPDH (data not shown). This is in keeping withthe fact that TAg, along with other transcription factors, inducesexpression of VP1 mRNA. Furthermore, in all studies, our AONs #2, #3, #4and #5 reduced VP1 RNA expression levels, along with striking reductionsin VP1 protein (see FIG. 5 , right panel).

We also tested whether a combination therapy of AONs #2 and #4 couldmore effectively reduce TAg and VP1 RNA levels. This combination wasalso selected based on the fact that the aforementioned Western blot forVP1 in AON-treated cells (FIG. 5 ) suggested that these two AONs lead tothe most potent loss of VP1 protein. Based on TAg and VP1 mRNAexpression levels, this combinatorial treatment did not yield evidencethat suggested that together they were more efficacious (FIG. 4 ).

The observed reduction in VP1 shows that TAg splice-targeting AONs areeffective. By reducing TAg RNA (and potentially protein expression),expression levels of the BKPyV late region genes and correspondingproteins are reduced. Moreover, alongside a role for VP1 inencapsulating the viral DNA, VP1 also serves a pivotal mediating role inthe infectivity of newly-formed viral particles by binding to the cellsurface of neighbouring and/or distant cells at sialic acids on glycans(Helle, F. et. al., Viruses, 2017). As such, the infectivity of BKPyVwould likely be compromised upon a reduction (or in the absence) of VP1protein.

TAg Splice-Targeting AONs Decrease BK Viral Titer

Concomitant with our screens for TAg and VP1 RNA and protein levels (atday 7) in HK2 cells pre-treated with our TAg splice-targeting AONs, wealso assessed the viral load in the culture supernatant at 3, 5 and 7days after BKPyV infection. We determined whether the decrease in VP1affected encapsulated viral DNA production, as a reduction in TAgexpression could potentially impact both viral genomic replication andVP1 protein generation. We determined the virus particles in culturesupernatant by quantitating encapsulated DNA. To discern betweenencapsulated and non-encapsulated DNA, we applied an (endo)nucleasetreatment to digest non-encapsulated DNA. As shown in FIG. 6 , thesestudies revealed that AON #1 at day 3 reduced viral DNA levels, but by 7days that this level has normalized and is similar to viral DNA levelsin scramble AON-treated cells (FIG. 6 ). AONs #2, #3 and #4 arecharacterized by reductions in viral titer at both time intervals, withAONs #2, #3 and #4 in particular attenuating encapsulated viral DNA upto 6-fold (FIG. 6 ). The aforementioned combination of AON #2 and #4only slightly reduced viral load at days 3 and 7 (FIG. 6 ).

Thus AON-mediated attenuation of TAg and VP1 RNA and protein leads to adecrease in virus production.

Alkyl Modifications at the 2′ Position of the Ribose Sugar

Altering the 2′-position of the ribose sugar on AONs impacts theircapacity to reduce TAg and VP1 RNA and protein levels, and BKV DNAproduction (FIG. 7 ). The aforementioned data is based on a 2′-O methyl(2′-OMe) modification of the ribose sugar on each nucleotide within anantisense oligonucleotide. RNA and protein have been harvested from HK2cells pre-treated with both 2′-OMe or 2′-methoxy (2′-MOE) nucleotides(see FIG. 7 ).

TAg Splice-Targeting AONs for Other Polyomaviruses

Alongside BKV, we have also developed AONs that similarly target TAg forJC virus (JCV). JCV has 75% sequence similarity to BKV, a level ofconservation that is also observed at the exon 1 – intron junction,whereas the sequence similarity at the intron - exon 2 junction isvirtually 100% (FIG. 8 ). AONs targeting exon 2 for BKV can thus alsoreduce JCV load. JCV can also infect kidney cells such as proximaltubule cells, which are believed to be a secondary site of infection,following initial infection via the tonsils and/or digestive tract. TheTAg splice-targeting AONs, in particular those targeting the intron -exon 2 splice site can thus simultaneously abrogate BKV and JCVproduction. Moreover, we are generating novel AONs that target theunique JCV exon 1 - intron sequence.

Given that the genomic sequence at the exon 1 – intron and intron – exon2 junctions for TAg have been determined for all known polyomaviruses,it is possible to design AONs that affect splicing of TAg in all ofthese polyomaviruses (FIG. 9 ).

Examples of suitable AON for other polyomaviruses are depicted in FIG. 9.

Example 2 Material and Methods Phylogenetic Conservation of BKV Subtypes

Complete genomic sequences of BK polyomavirus isolates were downloadedfrom the publicly available NCBI database (before Jul. 09, 2018). Fromthese records, only the isolates reporting a complete genome were usedfor analysis. Isolates “MM” and “FNL-9” were removed due to a largedeletion in the intron or duplication overlapping the acceptor splicesite respectively. Identical sequences were removed, yielding 248 uniquegenomic sequences of which the accession numbers are provided below:

AB211369, AB211370, AB211371, AB211372, AB211373, AB211374, AB211375,AB211376, AB211377, AB211378, AB211379, AB211381, AB211382, AB211383,AB211384, AB211385, AB211386, AB211387, AB211388, AB211389, AB211390,AB211391, AB213487, AB217917, AB217918, AB217919, AB217920, AB217921,AB260028, AB260029, AB260030, AB260031, AB260032, AB260033, AB263912,AB263913, AB263914, AB263915, AB263916, AB263917, AB263918, AB263919,AB263920, AB263921, AB263922, AB263923, AB263924, AB263925, AB263926,AB263927, AB263928, AB263929, AB263930, AB263931, AB263932, AB263934,AB263935, AB263936, AB263938, AB269825, AB269826, AB269827, AB269828,AB269829, AB269830, AB269831, AB269832, AB269834, AB269836, AB269837,AB269838, AB269840, AB269841, AB269842, AB269843, AB269844, AB269845,AB269846, AB269847, AB269848, AB269849, AB269850, AB269851, AB269852,AB269853, AB269854, AB269855, AB269856, AB269857, AB269858, AB269859,AB269860, AB269861, AB269862, AB269863, AB269864, AB269865, AB269866,AB269867, AB269868, AB269869, AB298941, AB298942, AB298945, AB298946,AB298947, AB301086, AB301087, AB301089, AB301090, AB301091, AB301092,AB301093, AB301094, AB301095, AB301096, AB301097, AB301099, AB301100,AB301101, AB365130, AB365132, AB365133, AB365134, AB365136, AB365137,AB365138, AB365139, AB365140, AB365141, AB365142, AB365144, AB365145,AB365146, AB365148, AB365149, AB365150, AB365151, AB365153, AB365154,AB365156, AB365157, AB365158, AB365159, AB365160, AB365162, AB365164,AB365165, AB365166, AB365167, AB365168, AB365170, AB365173, AB365174,AB365175, AB365176, AB365178, AB369087, AB369088, AB369089, AB369090,AB369092, AB369093, AB369094, AB369095, AB369096, AB369097, AB369098,AB369099, AB369101, AB464953, AB464954, AB464956, AB464957, AB464958,AB464960, AB464961, AB464962, AB485695, AB485696, AB485697, AB485698,AB485699, AB485700, AB485701, AB485703, AB485704, AB485707, AB485709,AB485710, AB485711, AB485712, AY628224, AY628225, AY628226, AY628227,AY628228, AY628229, AY628230, AY628231, AY628232, AY628233, AY628234,AY628235, AY628236, AY628237, AY628238, DQ305492, EF376992, FR720308,FR720309, FR720310, FR720311, FR720312, FR720313, FR720315, FR720317,FR720318, FR720320, FR720321, JF894228, JN192431, JN192432, JN192433,JN192435, JN192437, JN192438, JN192439, JN192440, JQ713822, KF055891,KF055892, KF055893, KP412983, KP984526, KY114802, KY114803, KY132094,KY487998, LC029413, LC309239, LC309240, LT960370, M23122, MF358970,MF627830, MF627831, V01108.

Splice Site Conservation and Phylogenetic Trees

Whole-gene sequences of TAg, including intron sequences, were alignedusing Prank (v. 140603). Manual adjustments were made to the alignedsequences to adjust for imperfections when aligning deletions. Aphylogenetic tree was constructed using the Neighbor-Joining method(MEGA version 10.0.5) with bootstrapping (1000 replications) and theKimura 2-parameter model. The phylogenetic tree was further visualizedin R (“ggtree”) and sequence logos were constructed (“ggseqlogo”) forthe acceptor and donor splice sites to show nucleotide specificconservation between subtypes. Subtypes of sequences were determinedusing reference sequences described by Zhong et al (Zhong, J Gen Virol,2009).

Oligonucleotide Design

Antisense oligonucleotides were designed as described in EXAMPLE 1. Forin vivo studies, a 2′-MOE AON (HYB_01) without 5′ 6-FAM label was used.

Animals

Male C57BL6/J mice between 6 and 10 weeks of age were intravenouslyinjected with 40 mg/kg 2′-MOE AON without 5′ 6-FAM label or saline(volume of +- 100 uL corrected for body weight). Animals were sacrificedunder isoflurane anesthesia using venous exsanguination 24 afteradministration of AON or saline. Organs were removed and fixed informalin and paraffin embedding.

Cell Culture

Human kidney proximal tubular epithelial cells (HK2, ATCC®) weremaintained in Dulbecco’s Modified Eagle Medium:Nutrient Mixture F-12(Gibco) supplemented with 3,3′,5-Triiodo-L-thyronine sodium salt(Sigma-Aldrich), insulin-transferrin-selenium-ethanolamine (ITS-X;Sigma-Aldrich), human epidermal growth factor (EGF; Sigma-Aldrich),hydrocortison (Sigma-Aldrich), and 100 U/mL penicillin-streptomycin(Gibco). Human renal proximal tubular epithelial cells (PTEC, SciencellResearch Laboratories) were maintained in complete REGM™ renalepithelial cell growth medium (Lonza). Primary human astrocytes(Sciencell Research Laboratories) were maintained in complete AstrocyteMedium (Sciencell Research Laboratorie). IPSc-derived astrocytes andoligodendrocytes were maintained in complete BrainPhys™ Neuronal Medium(Stemcell Technologies). All cells were cultivated at 37° C., 5% CO2.

AON Treatment and Viral Infection of Cells

Cells were seeded at the required cell density and cultivated overnight.Cellular uptake of AONs was achieved by cultivating cells in thepresence of 50 nM AON with lipofectamine 2000 for 4 h (human astrocytesand iPSc astrocytes/oligodendrocytes, Invitrogen) or lipofectamine 3000for 5 h (HK2 and PTEC, Invitrogen), after which the cells were washed innormal culture media. BKV infection of HK2 epithelial cells or humanrenal epithelial cells was performed as described in EXAMPLE 1. JCVinfection of astrocytes/oligodendrocytes was achieved by cultivating thecells in the presence of JC polyomavirus (MAD-4 strain, ATCC® VR-1583™)overnight. The cells were washed extensively after infection in order toremove excess viral particles. Culture media was partially refreshed,and supernatant samples were taken at specific time points afterinfection to study viral particle production. Re-infection of cells wasperformed by taking the supernatant of wells containing infected cellsafter treatment. This supernatant was then diluted 2-fold andtransferred to a new well containing uninfected, untreated cells for 2h, after which the cells were washed extensively. The infected cellswere washed after 7 days using 4% PFA.

Viral Load Determinations

Viral loads in the culture supernatant were performed as described inEXAMPLE 1, with the following exceptions. 1) 100 µL samples werecollected from every well at every time point. 2) Unpackaged DNA wasdegraded using the TURBO DNA-free kit (Invitrogen) before isolation.

Real-Time qPCR

Isolation of RNA, cDNA synthesis and real-time qPCR was performed asdescribed in EXAMPLE 1. However, after isolation of RNA, residual DNAwas degraded using the TURBO DNA-free kit (Invitrogen). For theamplification of T-antigen splice variants, the Phusion® High-FidelityPCR Kit was utilized using HF buffer and the following primers: forwardATGGAGCTCATGGACCTTTTAGG, reverse TGCAACTCTTGACTATGGGGG. QPCR detectionof JC virus RNA was performed using the following primers:

Gene Forward SEQ ID NO Reverse SEQ ID NO GADPH ACAACTTTGGTATCGTGGAAGG 40GCCATCACGCCACAGTTTC 41 TAg CACCCTGATAAAGGTGGGGAC 42GCAAAACAGGTCTTCATCCCAC 43 VP1 CCAAAGAATGCCACAGTGCAA 44GTGGGATCAGGAACCCAACAT 45

Antibodies

The following primary antibodies were used: rabbit anti-SV40 VP1(ab53977), mouse anti-SV40 T-antigen (PAb416), mouse anti-SV40 T-antigen(PAb108), rabbit anti-GAPDH (D16H11), biotinylated Lotus Lectin (LTL,B-1325). The rabbit anti-phosphorothioate antibody was kindly providedby Jonathan Watts (UMASS Medical School, MA, USA). The followingsecondary antibodies were used: goat-anti-rabbit Alexa 488 (A11008),goat-anti-rabbit Alexa 568 (A11011), goat-anti-rabbit HRP (P044801-2)and streptavidin Alexa 532 (S11224).

Protein quantificationProtein lysates were generated by lysing cells inlysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% SDS, 0.5%deoxycholate, 0.5% triton X-100 and protease inhibitors (pH 7.5). Sampleprotein concentrations were determined using Pierce™ BCA Protein AssayKit (Thermo Fisher Scientific). Quantification of protein expression wasperformed using the Wes Simple Western automated immunoassay system witha 12-230 kDa Separation Matrix and anti-Rabbit detection module(ProteinSimple).

Immunohistochemistry For re-infection experiments, cells were fixed with4% PFA and permeabilized in 0.3% Triton-X/3% BSA (Merck, Zwijndrecht,the Netherlands)/1% NGS (Dako, Amstelveen, Netherlands)/ 1% FCS in PBSfor 1 h at RT. Primary antibody was incubated in 3% BSA/1% NGS/1%FCS inPBS at 4° C. overnight, after which cells were washed extensively andincubated with secondary antibody for 1 h at RT. Image acquisition andquantification of re-infected cells was performed using the ImageXpressMicro High-Content Imaging System and MetaXpress software using custommodules to identify and count (TAg⁺) nuclei. Further processing of invivo images (colour deconvolution and thresholding) was performed usingImageJ.

Mouse organs were embedded in paraffin, cut and slides were dewaxed,rehydrated and endogenous peroxidases were quenched for 10 min at RT in3% H₂O₂ in methanol. Antigen retrieval was performed using Proteinase K(Agilent, Amstelveen, the Netherlands) for 10 min at RT, followed by ablocking step using Background buster (Innovex, Gujarat, India). Betweensteps, slides were washed in TBS/Tween. Primary antibody incubation(anti-phosphorothioate or LTL) was performed at 4° C. in 2%BSA/5%NGS inTBS/Tween. Secondary antibody incubation was performed for 90 min at RT.Nuclei were stained using Hoechst 33258 (Molecular Probes, Leiden, theNetherlands) and slides were mounted using Prolong Gold (Invitrogen).Image acquisition was performed using the Pannoramic MIDI II (3DHISTECH,Budapest, Hungary).

Next Generation Sequencing

RNA-seq was performed on RNA samples derived from infected, AON-treatedHK2 cells using Illumina sequencing technology. In short, sample qualitywas determined using the Fragment Analyzer and the NEBNext Ultra IIDirectional RNA Library Prep Kit for Illumina was used to process thesample(s). The sample preparation was performed according to theprotocol “NEBNext Ultra II Directional RNA Library Prep Kit forIllumina” (NEB #E7760S/L). Briefly, rRNA was depleted from total RNAusing the rRNA depletion kit (NEB#E6310). After fragmentation of therRNA reduced RNA, a cDNA synthesis was performed. This was used forligation with the sequencing adapters and PCR amplification of theresulting product. The quality and yield after sample preparation wasmeasured with the Fragment Analyzer. The size of the resulting productswas consistent with the expected size distribution (a broad peak between300-500 bp). Clustering and DNA sequencing using the NovaSeq6000 wasperformed according to manufacturer’s protocols. A concentration of 1.1nM of DNA was used. NovaSeq control software NCS v1.5 was used. Imageanalysis, base calling, and quality check was performed with theIllumina data analysis pipeline RTA3.3.5 and Bcl2fastq v2.20.

The human reference Homo_sapiens.GRCh38.dna.primary_assembly wascombined with the virus reference LC029411.1. The combined genome wasused for alignment of the reads for each sample. The reads were mappedto the reference sequence using a short read aligner based onBurrows—Wheeler Transform (Tophat v2.0.14) with default settings. Basedon the mapped locations in the alignment file the frequency of how oftena read was mapped on a transcript was determined with HTSeq v0.6.1p1.

Splice Event Identification Using Eventpointer

In order to identify alternative splicing events in NGS data,“Eventpointer” was applied on reads mapped to the viral referencegenome. The resulting splice events were quantified using Kallisto togenerate percent spliced (PSI) values for each event. For statisticaltesting, the scrambled AON was used as the control condition.

Pacific Biosciences Long-Read Sequencing

RNA integrity was first assessed on a bioanalyzer. The cDNA synthesiswas performed with the SMARTer cDNA synthesis kit (Takara) and specificlarge T products were amplified using Kapa HiFi HotStart Ready Mix(Roche). cDNA products were size selected after which amplicons werebarcoded per sample using the SMRTbell Barcoded Adapter Complete PrepKit (PacBio), then pooled equimolar and sequenced on a PacBio Sequel 1Mv3 LR SMRT cell.

The identification, polishing, and annotation of transcripts was carriedout using the Iso-Seq3 bioinformatics pipeline made public by PacificBiosciences (https://github.com/PacificBiosciences/IsoSeq3). Reads werefirst classified into full-length and non-full-length based on thepresence of sample-specific barcodes. To find transcript clusters, anisoform-level clustering algorithm (ICE) performs a pairwise alignmentand reiterative assignment of full-length reads to clusters based onlikelihood. After ICE, partial reads are added to the isoform clustersto increase coverage for a final consensus using the Arrow algorithm.The output from the bioinformatics pipeline is a set of full-lengthtranscript sequences that can be mapped to the reference sequence toconstruct an annotation file in GFF format. Based on the Arrowalgorithm’s predicted consensus accuracy, transcript sequences that hada predicted accuracy of > 99% (excluding QVs from the first 100 bp andlast 30 bp due to occasionally insufficient coverage for accurateestimation of accuracies) were considered HQ transcripts and used forfurther analysis. The HQ transcript sequences were mapped back to thereference sequence and filtered for > 99% alignment coverage and > 85%alignment identity. Redundant transcripts were collapsed to create afinal dataset used in this study.

Results Development of Novel BKV-Targeting AON

As shown in FIGS. 10 and 11 , alongside our previous AONs, we elected todesign 9 new AONs that target BKV TAg, 6 of which target the exon 1 –intron portion (designated HYB_06, _07, _08, _09, _10, and _11), while 2new AONs target the exon 2 - intron portion (termed HYB_12 and _13).Alongside these 9 new TAg exon-intron junction targeting AONs, we alsotested 2 AONs previously described by Santaris Pharma (WO2012/143427A1),which have different compositions relative to our AONs yet arecomplementary to a part of the exon 1-intron junction (SAN_74) or solelya part of exon 2 (SAN_73). Furthermore, we also designed and tested anAON that binds exclusively to the coding region of exon 1, namelyHYB_14. The AONs progressively shift from primarily exonic to includingsignificant intron-binding sequence for exon 1 targeting AONs, nowcollectively termed (HYB_01, _02, _03, _06, _07, _08, _09, _10, _11) andexon 2 targeting AONs (HYB _04, _05, _12 and _13).

AON-Mediated Reduction in BKV TAg RNA

The BKV-targeting AONs displayed varying ranges of potency in reducingTAg mRNA levels. As shown in FIGS. 12 and 19 , of the 14 AONs designedto target the exon-intron junction of TAg, HYB_01, HYB_03 and HYB_11induced the greatest reductions in TAg mRNA expression, generally in therange of 8- or greater-fold attenuation in the RNA levels of this viralDNA driver at 7 days post-infection.

Furthermore, our data also suggest that AONs targeting the exon 1 –intron junction is more effective in reducing TAg mRNA levels than AONstargeting the exon 2 - intronic junction (HYB_04, HYB_05, HYB_12 andHYB_13). This trend is bioinformatically depicted in FIGS. 18 and 25 ,where solely HYB_05 clusters with the AONs that target the exon 1 -intronic junction. Similarly, SAN_73 and SAN_74 proved largelyineffective in reducing TAg mRNA expression levels. Of note, thesereductions in TAg mRNA are observed in the setting a high MOI, namely inthe range of 100.

Alongside HK2 cells, we also tested our BKV-targeting AONs in primaryproximal tubule epithelial cells (hPTECs). Based on the significant andconsistent reductions in TAg mRNA expression levels observed in HK2cells with HYB_01, HYB_03 and HYB_11, we elected to proceed at thisphase with these three being designated our ‘lead compounds’. As shownin FIG. 28 , HYB_01, HYB_03 and HYB_11 all dramatically reducedexpression of TAg mRNA in hPTECs (n=3 biological replicates), whileHYB_14 proved ineffective in reducing TAg mRNA levels in hPTECs.

Interestingly, the majority of our studies involve pre-treatment withAON prior to infection with BKV. Preliminary studies in which we firstinfected HK2 cells with BKV and subsequently treated the cells with AON(namely HYB_01) revealed that our BKV-targeting AONs can efficientlyrepress BKV TAg expression in cells that harbour BKV 7 dayspost-infection (FIG. 26 , left panel), and that the administration ofmultiple dosages post-infection can potentially further suppress TAgmRNA expression levels (FIG. 26 , right panel).

AON-Mediated Reduction of VP1 RNA and Protein

Interestingly, VP1 mRNA expression levels were reduced by most of theBKV-targeting AONs, with the exception of HYB_04, HYB_12, HYB_13 andHYB_14 (FIGS. 13 and 20 ). Similar to our results obtained for TAg mRNA,HYB_01, HYB_03 and HYB_11 in particular resulted in striking reductionsin VP1 RNA expression in HK2 cells (FIG. 13 ). Moreover, HYB_01efficiently reduced VP1 mRNA levels regardless of whether the AON wasadministered prior to infection or post-infection with BK virus in HK2cells (FIG. 26 , left and right panels). Importantly, the observedreductions in VP1 mRNA resulted in dramatic attenuation of VP1 proteinlevels in HK2 cells (FIGS. 14-15 and 21-22 ). In keeping with ourobservations for TAg mRNA levels, SAN_73 and SAN_74 did notsignificantly impact expression levels of VP1 mRNA and protein.

As shown in FIGS. 29-31 , HYB_01, HYB_03 and HYB_11 all dramaticallyreduced expression of VP1 mRNA and protein in hPTECs (n=3 biologicalreplicates). Furthermore, HYB_14 did not affect VP1 mRNA and proteinlevels. Moreover, we have also gained preliminary evidence that our AONsalso effectively reduce VP3 protein levels (FIGS. 30 and 32 ).

Infection and Re-Infection of Human Proximal Tubule Epithelial Cells

We next assessed whether our broad assortment of BKV-targeting AONscould impact the degree of infection and re-infection by BKV in HK2cells. To achieve this, we first treated HK2 cells with BKV-targetingAONs and infected the cells with BKV. After 7 days, we harvested theviral particle-containing supernatant and used this to infect newbatches of untreated HK2 cells. After 7 days, we performedimmunofluorescent staining for TAg-infected cells and scored this as apercent positive (by counterstaining with Hoechst for nuclei). As shownin FIGS. 17 and 24 , these studies revealed that cells previouslytreated with our BKV-targeting AONs displayed significantly lower levelsof re-infection. In contrast, pre-treatment of HK2 cells with SAN_73 andSAN_74 did not decrease re-infection levels to the degree observed withHYB_01, HYB_03 and HYB_11.

BKV-Targeting AONs Influence Viral Particle Production

The observed reductions in VP1 protein, a protein that is required topackage the BK virus DNA, should severely impact the formation andrelease of new viral particles into the supernatant. Indeed, as shown inFIGS. 16, 23 and 33 , the vast majority of our BKV-targeting AONsdecrease viral particle production 7 days post-infection, whereby thegreatest reductions are yielded by HYB_01, HYB_03 and HYB_11. Incontrast, SAN_73 and SAN_74 were found to but slightly decrease viralparticle production. This mechanism is likely responsible for decreased(re-)infection of local and distal cells in vitro, and strongly suggeststhat the uptake of our BKV-targeting AONs in proximal tubule epithelialcells of the kidney post-kidney transplantation would be an effectivetherapeutic modality in preventing BKV activation and/or spreading.

BKV-Targeting AONs Modulate Splicing of TAg

To gain mechanistic insight into how our BKV-targeting AONs are leadingto the herein described reductions in TAg and VP1 mRNA, we performedRNA-seq of RNA harvested from HK2 cells that were treated with ascrambled AON, HYB_01, HYB_03, HYB_11, HYB_14, SAN_73 or SAN_74, afterwhich the cells were infected with BKV for 2 hours. Post-washing, thecells were cultured for 7 days after which RNA was harvested, assessedon a bioanalyzer for signs of degradation. Subsequently, equivalentquantities of RNA were ribo-depleted, underwent library preparationafter which RNA-seq was performed. In keeping with our aforementionedreductions in TAg and VP1 mRNA in HK2 cells, coverage of the BKV genomefrom scrambled control, HYB_14 or SAN_73 and SAN_74-treated cells wereclearly higher than those treated with BKV-targeting AONs (FIG. 42 ).For analysis of changes in BKV splicing, both the human genome as wellas the BKV genome was provided for alignment of sequences, with Bowtiebeing used to align the paired-end reads.

In order to quantify alternative splicing in the samples, EventPointerwas applied. A specific GTF file with different transcripts of the viruswas used. The algorithm tries to identify possible alternative splicingevents and relate each of the transcripts to the possible alternativepaths. To assess splicing in a highly complex pre-mRNA such as TAg, thepre-mRNA was dissected into unique splice events, leading the TAgpre-mRNA to initially be separated into 7 fragments. At each junction,defined by the frequency that a splice event was detected, the percentspliced in (PSI) was determined. This resulted in four uniquealternative splice events that occured in all conditions at a highfrequency as determined by EventPointer. The frequency of these eventswas scored using Kallisto software, resulting in a quantification pertranscript (with units being transcripts per million). After statisticaltesting for significance, significant changes in splicing were observedat the exon 1 - intron junction of TAg, precisely the site where ourAONs are binding and predicted to impact splicing (FIG. 35 ). Here, theupper panel provides a schematic of the relevant splicing events in theTAg pre-mRNA that can subsequently be dissected into unique splicingevents as shown in the bottom panel. For splice event 1, HYB_01 yields ahighly significant modulation in the splicing pattern for truncated Tantigen (P=0.0108), as an alternative splice acceptor site ispreferentially used relative to HK2 cells treated with a scrambledcontrol AON (Z-value = 2.5484). Similarly, for splice event 2 a highlysignificant change in splicing is detected (p=0.0149) where HYB_01 leadsto the preferential generation of small t antigen (tAg) as opposed toTAg (Z-value -2.4342). In contrast, no significant splicing changes aredetected in cells treated with HYB_14, SAN_73 or SAN_74. The absence ofsplicing modulation for SAN_74 is in particular striking given that thisAON also bridges the exon 1 - intron junction. This strongly suggeststhat the size of our AONs (20 nt as opposed to 16 nt in length) plays arole in determining their capacity to impact splicing of TAg,potentially as a result of steric hinderance.

Furthermore, the data depicted in FIG. 35 also suggest that themodulation of splicing at the exon 1 - intronic junction (splice event2) influences splicing decisions occurring within exon 2 (splice event1). Hence, our BKV-targeting AONs appear to be triggering a mutuallyexclusive or complex splicing event downstream in the TAg pre-mRNA.These studies are based on a biological n=3.

Supporting evidence that our BKV-targeting AONs mediate changes in TAgsplicing were obtained by performing long-range PCR using high-fidelityPhusion polymerase. As shown in FIG. 43 , dramatic shifts in truncated Tantigen acceptor site usage (splice event 1 in FIG. 35 ) were observedfor HYB_01 and HYB_11 as compared to scramble control-treated cells,with a more subtle shift evident for HYB_03. These data confirm thatAONs targeting the exon 1 -intron junction profoundly impact splicingdecisions within exon 2.

To gain additional insight into the splice-mediating effects of our AONson BKV TAg, we also employed PacBio sequencing to generate long-sequencereads of TAg, where primers binding to the 5′- and 3′-ends of TAg wereused to amplify full-length TAg pre-mRNAs. These studies would yieldprecise insight into the exact usage of individual splice sites withinTAg, as well as potential mutually exclusive or complex events asindicated in our RNA-seq data. RNA degradation was assessed on abioanalyzer, and following target enrichment by PCR, the PCR productswere size selected. The cDNA library was prepared, ends repaired,adapters ligated, DNA purified and SMRTbell DNA sequenced. Subsequently,the subreads were converted into circular consensus reads (insertsequence reads). As shown in FIG. 36 , in keeping with our RNA-seq data,these studies indicate that AONs directed at the TAg exon 1 - intronacceptor impacts usage or access to this splice site. Our BKV-targetingAONs, in particular HYB_01, reduces levels of TAg produced (left panel;left bars), leading to a shift towards increased levels of small tantigen and unspliced large T antigen (left panel; middle and rightbars, respectively). Furthermore, the data provide further support forAON-mediated modulation of truncated T antigen splicing (right panel).

It is important to note that the herein displayed efficacy of ourBKV-targeting AONs in modulating TAg splicing at the exon 1 - intronjunction could lead to the usage of (alternative) cryptic splice donorsites. The potential use of either an upstream (coding sequence portionof exon 1) or downstream cryptic splice site (intronic portion prior toexon 2) could lead to frameshifted mRNAs that generally lead to theintroduction of premature termination codons. It is well establishedthat these aberrant transcripts would rapidly be degraded within thecell by nonsense-mediated decay (Hug, N., et al., Nucleic AcidsResearch, 2016). Importantly, this rapid processing would likelypreclude us from detecting the majority of these malformed transcripts.

Nevertheless, our quantitative and qualitative analyses of the remainingTAg transcripts clearly indicates that our BKV-targeting AONs elicitstriking reductions in TAg mRNA levels and simultaneously impact thebalance of mRNAs formed as a result of pre-mRNA splicing. These dataimplicate the dual modulation of TAg expression and splicing as a potentmeans of attenuating BKV particle production and infectibility.

In Vivo Uptake of BKV-Targeting AONs

Our data generated in vitro for BKV-targeting AONs have been chemicallymodified to contain a 2′-O methyl (2′-OMe) modification of the ribosesugar on each nucleotide within an antisense oligonucleotide.Importantly, the uptake of AONs in vivo has consistently been found tobe markedly improved if the 2′ hydroxy group is replaced with a2′-methoxy (2″-MOE) group. Hence, we modified HYB_01, our lead compoundto possess both the complete phosphorothioate backbone and 2′-MOEgroups, and injected this AON intravenously via the tail vein intoC57BL/6J mice. At 24 hours post-injection the mice were sacrificed andthe kidney, liver, spleen, brain and muscle harvested and sectioned. Asshown in the immunohistochemical staining in FIG. 44 and FIG. 45 ,HYB_01 displayed excellent uptake in the kidney cortex, and inparticular in the proximal tubule epithelial cells of the kidney (asevidenced by uptake in lotus tetragonolobus lectin (LTL)-positive cellsof the kidney). Moreover, HYB_01 was also detectable in Kupffer cells ofthe liver, and undetectable in the brain (see FIG. 45 ). Furthermore,the AONs were detectable in the white pulp of the spleen, minimallydetectable in the heart and undetectable in muscle (data not shown).

TAg Splice-Targeting AONs for Other Polyomaviruses

Albeit that JC virus (JCV) is well established to infect the proximaltubule epithelial cells of human kidneys, our repeated attempts toachieve this were unsuccessful. Therefore, we elected to infect otherhuman cells that are known to be susceptible to JC virus and play a rolein the development of JCV-related pathophysiologies, namely astrocytes.For these studies, we pre-treated either human induced pluripotent stemcell-derived astrocytes or a human primary astrocytic cell line with oneof our 5 JCV-targeting AONs (FIG. 37 ), namely HYB_15-19 (for 4 hours ata concentration of 50nM per AON), and subsequently infected the cellswith JCV overnight with a titer of 10^(4.5) TCID₅₀/0.2 mL (informationprovided by supplier based on infection of Cos-7 cells at 7 dayspost-infection). As shown in FIGS. 38 and 39 , in keeping with ourobservation that targeting the exon 1 - intron junction of BKV TAgdiminished TAg and VP1 mRNA expression levels, HYB_15, HYB_16 and HYB_17resulted in marked reductions in both JCV TAg and VP1 mRNA expressionlevels in iPS cell-derived astrocytes (n=1). Furthermore, in primaryhuman astrocytes we also observed striking reductions in JCV TAg and VP1mRNA expression levels at varying titers of JCV administration (FIGS. 40and 41 ; n=2).

TABLE Human Polyomavirus genus Virus name NCBI ref seq Clinicalcorrelate (if any) 1 Beta BK polyomavirus NC_001538 Py-assoc.nephropathy; haemorrhagic cystitis 2 Beta JC polyomavirus NC_001699Progressive multifocal leukoencephalopathy 3 Beta KI polyomavirusNC_009238 4 Beta WU polyomavirus NC_009539 5 Alpha Merkel cellpolyomavirus NC_010277 Merkel cell cancer 6 Delta Human polyomavirus 6NC_014406 HPyV6 assoc. pruritic and dyskeratotic dermatosis 7 DeltaHuman polyomavirus 7 NC_014407 HPyV7-related epithelial hyperplasia 8Alpha Trichodysplasia spinulosa polyomavirus NC_014361 Trichodysplasiaspinulosa 9 Alpha Human polyomavirus 9 NC_015150 10 Delta MWpolyomavirus NC_018102 11 Delta STL polyomavirus NC_020106 12 AlphaHuman polyomavirus 12 NC_020890 13 Alpha New Jersey polyomavirusNC_024118 *source Wikipedia.

1-14. (canceled)
 15. An antisense oligonucleotide of 12 to 30nucleobases in length comprising a sequence that is a reverse complementof a contiguous stretch of at least 12 nucleobases of a polyomavirusT-antigen pre-mRNA, wherein the antisense oligonucleotide is capable ofmodulating splicing of said T-antigen pre-mRNA in a cell, and whereinthe antisense oligonucleotide is directed towards the exon 1 - intronjunction of the polyomavirus T-antigen pre-mRNA.
 16. The antisenseoligonucleotide of claim 15, comprising at least 12 contiguousnucleobases of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, or a variant thereof, wherein the variant comprises one or moresubstitutions by a nucleobase analogue having the same base pairingspecificity as the replaced nucleobase.
 17. The antisenseoligonucleotide of claim 15, wherein the antisense oligonucleotide is asingle stranded RNA antisense oligonucleotide.
 18. The antisenseoligonucleotide of claim 15, wherein the antisense oligonucleotidecomprises a modification capable of rendering an RNA duplex resistant tothe action of RNase H, and wherein the RNA duplex comprises theoligonucleotide and an oligonucleotide complementary thereto.
 19. Theantisense oligonucleotide of claim 18, wherein the modificationcomprises a 2′ sugar modification.
 20. The antisense oligonucleotide ofclaim 19, wherein the 2′ sugar modification is a 2′- alkoxy,2′-alkoxyalkoxy, 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE),2′-S-constrained-ethyl (2′-cEt) or locked nucleic acid (LNA)modification.
 21. The antisense oligonucleotide of claim 15, furthercomprising at least one backbone modification.
 22. The antisenseoligonucleotide of claim 21, wherein the backbone modification is aphosphorothioate, 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE),2′-S-constrained-ethyl (2′-cEt), locked nucleic acid (LNA), peptidenucleic acid (PNA) or morpholino (PMO) modification.
 23. The antisenseoligonucleotide of claim 15, comprising at least 17 contiguousnucleobases of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, or a variant thereof having one nucleobase substitution between thefirst and the last nucleobase of the at least 17 contiguous nucleobases.24. The antisense oligonucleotide of claim 15, comprising at least 12contiguous nucleobases of SEQ ID NO: 3, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or a variant thereof,wherein the variant comprises one or more substitutions by a nucleobaseanalogue having the same base pairing specificity as the replacednucleobase.
 25. The antisense oligonucleotide of claim 15, comprising atleast 12 contiguous nucleobases of SEQ ID NO: 23, SEQ ID NO: 24, or avariant thereof, wherein the variant comprises one or more substitutionsby a nucleobase analogue having the same base pairing specificity as thereplaced nucleobase.